DNA constructs and viral vectors comprising a smooth muscle promoter

ABSTRACT

Disclosed is a smooth muscle cell specific promoter, the SM22α gene promoter as well as the murine cDNA and genomic SM22α nucleic acid sequences. Also disclosed are methods of preventing restenosis following balloon angioplasty and methods of treating asthma based on inhibition of smooth muscle cell proliferation by expressing cell cycle control genes, or contraction inhibiting peptides in smooth muscle cells, under the control of the SM22α promoter.

The government owns rights in the present invention pursuant to grantnumbers R01-HL48257, U01 AI34566 and R01HL51145 from the Public HealthService.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of geneexpression, particularly tissue specific expression, and moreparticularly smooth muscle cell specific expression. The invention alsorelates to cell proliferation diseases such as atherosclerosis,restenosis following balloon angioplasty and airway blockage in asthma.

2. Description of the Related Art

The phenotypic plasticity of smooth muscle cells (SMCs) permits thismuscle cell lineage to subserve diverse functions in multiple tissuesincluding the arterial wall, uterus, respiratory, urinary and digestivetracts. In contrast to fast and slow skeletal muscle cells which fuseand terminally differentiate before expressing contractile proteinisoforms, SMCs are capable of simultaneously proliferating andexpressing a set of lineage-restricted proteins including myofibrillarisoforms, cell surface receptors and SMC-restricted enzymes. Moreover,in response to specific physiological and pathophysiological stimuli,SMCs can modulate their phenotype by down-regulating a set ofcontractile protein genes, and in so doing, convert from the so called"contractile phenotype" to a de-differentiated "secretory phenotype"(Mosse et al., 1985; Owens et al., 1986; Rovner et al., 1986; Taubman etal, 1987; Ueki et al., 1987; Belkin et al., 1988; Glukhova et al., 1988;Chaponnier et al., 1990; Gimona et al., 1990; Shanahan et al., 1993).

This phenotypic modulation has been implicated in the pathogenesis of anumber of disease states including atherosclerosis and restenosisfollowing coronary balloon angioplasty (Ross, 1986; Schwartz et al.,1986; Zanellato et al., 1990; Ross, 1993; Olson and Klein, 1994) and mayalso contribute to the airway remodeling seen in asthma (James et al.,1989). Restenosis following coronary balloon angioplasty is a majorproblem, and contributes to the 40% failure rate of this procedure(Schwartz, et al., 1992; Liu, et al., 1989). Restenosis occurs becausethe smooth muscle cells are stimulated to proliferate after angioplastyand thus block the arterial wall. Because of restenosis, balloonangioplasty is used mainly for palliation in patients who are notacceptable candidates for open heart surgery (Scientific AmericanMedicine, Rubenstein and Federman, Eds., March 1993, Section 1, XII,page 11). A method is needed, therefore, to control or inhibit theproliferation of smooth muscle cells after angioplasty.

In addition, ample evidence demonstrates that airway smooth musclecontraction plays a critical role during acute episodic airflowobstruction in asthma (Knox, 1994; Rodger, 1992; Pueringer andHunninghake, 1992; Black, 1991). Extra-muscular factors, includingsubmucosal thickening (James et al., 1989), vascular engorgement(Lockhart et al., 1992), periadventitial inflammation (Ingram, 1991), orpersistent airway closure with bronchial non-reopening (Gaver et al.,1990), may amplify lumenal narrowing during bronchial smooth muscleconstriction. While these factors exacerbate airflow obstruction, itremains airway smooth muscle contraction that is ultimately responsiblefor the acute decrement of airway caliber. Prevention or reversal ofmuscular bronchoconstriction has therefore acquired a prominent role inasthma treatment. Because they inhibit force generation by airway smoothmuscle, β₂ -adrenergic agonists are recommended in recent NIH guidelinesas "the medication of choice for treatment of acute exacerbations ofasthma . . . " (National Asthma Education Program, 1991).

Yet, despite their obvious clinical utility, β₂ -adrenergic agonists arenot ideal medicines. Their chronic use has been associated withdiminished control of asthma symptoms, due perhaps to receptordown-regulation (Tashkin et al., 1982), to enhanced constrictorhyperresponsiveness following cessation of regular β₂ -adrenergicagonist use (Vathenen et al., 1988), or simply to masking of theunderlying inflammatory process. Though controversial (Wanner, 1995),chronic use of potent β₂ -adrenergic agonists might even increase asthmamortality (Crane et al., 1989). Furthermore, wide clinical andlaboratory experience (Rossing et al., 1982) demonstrates that inhaledβ₂ -adrenergic agonists do not fully prevent acute airway narrowing inresponse to provocative stimuli. Together, these accumulated dataindicate that: 1) inhibition of airway smooth muscle contraction doesrepresent an important facet of the treatment of asthma, but 2) use ofβ₂ -adrenergic agonists alone to achieve this goal is not the optimalsolution.

Relatively little is understood about the molecular mechanisms thatcontrol SMC-specific gene expression. Only three smooth muscle cellspecific genes have been studied intensively throughout development,SMα-actin, SM-myosin heavy chain and calponin-h1. However, of thesethree, SMα-actin and calponin-h1 are expressed in various tissues otherthan smooth muscle. It is also unfortunate that all three of the smoothmuscle genes, SMα-actin, SM-myosin heavy chain and calponin-h1 are onlyexpressed in quiescent vascular smooth muscle cells, and not inproliferating cells. Thus, there is still a need for discovery of asmooth muscle cell specific promoter that is not expressed in othertypes of cells and is constitutively expressed in both quiescent andproliferating cells.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacks in theprior art by providing a promoter specific for expression in smoothmuscle cells, and offering the further advantage that the control ofexpression directed by the promoter is constitutive and cell cycleindependent. The promoter of the present invention thus promotestranscription in both resting and proliferating cells, in contrast toother known smooth muscle cell promoters that are down-regulated inproliferating cells. This promoter may be used therefore, to expressheterologous proteins or mRNA's in proliferating smooth muscle cells andto control proliferative diseases or to promote angiogenesis, forexample.

The invention may be described, in certain embodiments, as an isolatednucleic acid segment comprising an SM22α promoter sequence. The isolatedSM22α promoter may be described as the region immediately upstream ofthe translational start site of the murine SM22α gene. As describedherein a nucleic acid segment having a sequence according to bases899-1382 of SEQ ID NO:1, is also effective to promote transcription in asmooth muscle cell and a nucleic acid segment having that sequence orone that is hybridizable to that sequence under high stringencyconditions and further is effective to promote transcription of aheterologous gene in a smooth muscle cell would also fall within thescope of the claimed invention. Such homologous promoters may beisolated from an animal sequence, such as from a mouse, pig, rat,hamster, rabbit or and even a human genome or cDNA library using any ofthe sequences disclosed herein as a molecular probe. In addition, basedon the present disclosure, one of skill might construct such a promoterby splicing elements taken from various sources including, but notlimited to, chemically synthesized nucleic acid molecules, or elementsremoved from other naturally occurring promoters. It is understood thatany such promoter, or a promoter having the essential elements of thepromoter disclosed herein would be encompassed by the spirit and scopeof the invention claimed herein.

The promoter region of the present invention may be defined ascomprising that region of the genome immediately upstream (5') of thestructural SM22α gene, and controlling expression of that gene. Forexample, the promoter may comprise the region of up to 30, 40, 50, 100,500, 1,000, 1,500, 2,000 or even up to 5,000 bases directly upstream ofthe translational start site of the SM22α gene, and more specifically,an SM22α promoter of the present invention may be described as anisolated nucleic acid segment that comprises a contiguous sequence ofbases 1-1381 (-1338 to +41) of SEQ ID NO:1. The designations of -1338 to+41 and the like indicate the position of a base relative to thetranscriptional start site (+1), which, in the murine genome, isdisclosed herein to be base 1341 of SEQ ID NO:1. The promoter of thepresent invention may also be described as an isolated nucleic acidsegment that comprises a contiguous sequence of bases 899-1381 (-441 to+41) of SEQ ID NO:1. Certain elements of the promoter that areidentified in light of the present disclosure are a TATA box 29-bp 5' ofthe start site, five consensus E boxes/bHLH myogenic transcriptionfactor binding sites located at bps -534, -577, -865, -898, -910, and-1267, three consensus GATA-4 binding sites located at bps -504, -828,-976, two AT-rich, potential MEF-2/rSRF binding sites located at bps-407 and -770 and at least one cis-acting, positive transcriptionalregulatory element contained by bp -435 to -416. In addition, thepromoter of the present invention contains consensus CArG/SRF bindingsites located at bps -150 and -273, one CACC box located at bp -104 andtwo potential zeste-binding sites, 5' ends at bp -435 and -421,respectively, which are illustrated in FIG. 11, SEQ ID NO:20.

Thus, the promoter of the present invention may comprise some or all ofthe elements described in the previous paragraph. Such elements may beisolated and recombined by techniques well known in the art to produce asmooth muscle cell specific promoter that may be smaller than the 441 to482 bases disclosed herein as a minimal sequence required forconstitutive smooth muscle cell transcription. It is also known thatcertain stretches of sequence in the promoter are required for spacingof the cis acting elements and that any sequence that does not imparthairpin loops or other deleterious structural properties may besubstituted for those regions so long as the spacing remains the same.It is understood that all such promoters would be encompassed by thepresent invention.

The isolated nucleic acid segments of the present invention may also bedefined as comprising a nucleic acid sequence or even a gene operativelylinked to an isolated SM22α promoter sequence. Operatively linked isunderstood to mean that the gene is joined to the promoter region suchthat the promoter is oriented 5' to the gene and is of an appropriatedistance from the transcription start site, so that the transcription ofthe gene will be dependent on or controlled by the promoter sequence.The arts of restriction enzyme digestion and nucleic acid ligation to beused in construction of a promoter-gene construct are well known in theart as exemplified by Maniatis et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor, N.Y., 1982, (incorporated herein byreference). Therefore one would, using standard techniques, prepare agene by restriction enzyme digestion to have a compatible end sequence,or even a blunt end, to be ligated downstream of the SM22α promoter. Therestriction enzyme recognition site may be a naturally occurringsequence, or a sequence generated by site directed mutagenesis, by aPCR™ primer sequence or by any other means known in the art.Alternatively, one might chemically synthesize a gene or gene fragmentor an oligonucleotide containing an appropriate restriction enzymerecognition sequence or one might prepare a gene by any of severalmethods known in the art.

The gene or nucleic acid segment may be, for example, a structural genethat encodes a full length protein, a portion or part of a protein, or apeptide that one desires to express in a smooth muscle cell. The genemay also encode an RNA sequence, such as an antisense oligonucleotidesequence, or even a regulatory sequence that affects the expression ofanother gene or genes. In certain preferred embodiments of theinvention, the gene will be a cell cycle control gene, such as aretinoblastoma (Rb) gene, p53, a cell cycle dependent kinase, a CDKkinase, a cyclin, a cell cycle regulatory protein, an angiogenesis genesuch as VEGF, or any other gene, the expression of which will affectproliferation of the smooth muscle cells in which the gene is expressed,or will effect the growth of new blood vessels. Alternatively, thenucleic acid segment may encode an antisense RNA effective to inhibitexpression of a cell cycle control gene or regulatory element. Antisenseconstructs are oligo- or polynucleotides comprising complementarynucleotides to the control regions or coding segments of a DNA molecule,such as a gene or cDNA. Such constructs may include antisense versionsof both the promoter and other control regions, exons, introns andexon:intron boundaries of a gene. Antisense molecules are designed toinhibit the transcription, translation or both, of a given gene orconstruct, such that the levels of the resultant protein product arereduced or diminished. Antisense RNA constructs, or DNA encoding suchantisense RNAs, may be employed to inhibit gene transcription ortranslation or both within a host cell, either in vitro or in vivo, suchas within a host animal, including a human subject. Of course, theantisense constructs have evident utility in the types of nucleic acidhybridization described herein. The gene may also encode an antigenicsequence and the necessary leader sequence for transport to the cellsurface, or it may encode an enzyme, or an intracellular signal proteinor peptide, or it may even encode an SM22α gene or SM22α cDNA gene.Particularly preferred is a constitutively active form of the Rb geneproduct that inhibits cellular proliferation, disclosed in Chang et al.,1995 (incorporated herein by reference).

The present invention may also be described, in certain embodiments, asa recombinant vector that is capable of replication in an appropriatehost cell and that comprises an SM22α promoter sequence as disclosedherein, including an SM22α promoter operatively linked to a gene ornucleic acid segment. Preferred vectors include, but are not limited to,a plasmid, a raus sarcoma virus (RSV) vector, a p21 viral vector or anadenoviral vector. In addition, a variety of viral vectors, such asretroviral vectors, herpes simplex virus (U.S. Pat. No. 5,288,641,incorporated herein by reference), cytomegalovirus, and the like may beemployed, as described by Miller (1992, incorporated herein byreference). Recombinant adeno-associated virus (AAV) and AAV vectors mayalso be employed, such as those described in U.S. Pat. No. 5,139,941,incorporated herein by reference. Recombinant adenoviral vectors arecurrently preferred. Techniques for preparing replication-defectiveinfective viruses are well known in the art, as exemplified byGhosh-Choudhury & Graham (1987); McGrory et al. (1988); and Gluzman etal. (1982), each incorporated herein by reference. Also preferred areplasmid vectors designed for increased expression such as thosedescribed in Tripathy et al., 1996.

A preferred adenovirus used in the practice of the present invention isreplication-defective. A preferred replication-defective adenovirus isone that lacks the early gene region E1 or the early gene regions E1 andE3. For example, the foreign DNA of interest, such as the SM22α promoterand gene of the present invention may be inserted into the region of thedeleted E1 and E3 regions of the adenoviral genome. In this way, theentire sequence is capable of being packaged into virions that cantransfer the foreign DNA into an injectable host cell. A preferredadenovirus is a type 5 adenovirus and a SM22α promoter and codingsequence are preferably flanked by adenovirus type 5 sequences.

In certain embodiments of the invention, the vector of the presentinvention is dispersed in a pharmaceutically acceptable solution.Preferred solutions include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one will desire topurify the vector sufficiently to render it essentially free ofundesirable contaminant, such as defective interfering adenovirusparticles or endotoxins and other pyrogens such that it will not causeany untoward reactions in the individual receiving the vector construct.A preferred means of purifying the vector involves the use of buoyantdensity gradients, such as cesium chloride gradient centrifugation.

The present invention may also be described, in certain embodiments, asa method of expressing a gene in a smooth muscle cell comprising thesteps of: obtaining an isolated nucleic acid segment comprising a geneoperatively linked to an SM22α promoter region; transferring thatnucleic acid segment into a smooth muscle cell; and maintaining thesmooth muscle cell under conditions effective to express the gene. Thegene may be a heterologous gene or the SM22α gene, for example. In thismethod of the invention, the SM22α promoter region preferably includesbases -441 to +41 of the SM22α gene (899-1382 of SEQ ID NO:1) or evenbases -441 to -1 of murine SM22α gene (900-1340 of SEQ ID NO:1) and mayinclude up to 5,000 bases of the SM22α promoter. In the practice of thismethod, the heterologous gene is preferably a reporter gene, a cellcycle control regulatory gene, an angiogenesis gene, an antisensemolecule, or it may encode a muscle contraction inhibiting peptide, andmay encode an Rb gene product or a peptide having the sequence MIRICRKK,SEQ ID NO:19. The Rb gene may be the wild type Rb gene or it may be analtered gene such that the gene product is phosphorylation deficient. Itis noteworthy that it may not be necessary to collect the gene productin the practice of the present method. For example, if the gene productis a cell cycle regulatory element, or a contraction inhibiting peptide,then the cell itself will be the target of that effect and the utilityof the method will not depend on collecting or even on identifying aprotein product. However, certain gene products will have utility asmarkers of gene expression and as useful proteins or peptides producedby a recombinant cell.

In addition, the present invention may be described as a method ofinhibiting smooth muscle cell proliferation comprising the steps of:obtaining an isolated nucleic acid segment comprising a cell cycleregulatory gene operatively linked to an SM22α promoter region;transferring the nucleic acid segment into a smooth muscle cell toobtain a transfected cell; and maintaining the smooth muscle cell underconditions effective to express the cell cycle regulatory gene; whereinexpression of the cell cycle regulatory gene inhibits proliferation ofthe smooth muscle cell. In the practice of the method, the cell cycleregulatory gene operatively linked to an SM22α promoter region maycomprise a viral vector, a plasmid vector or it may comprise anadenoviral vector. Further, the cell cycle regulatory gene maypreferably encode Rb, p53, cell cycle dependent kinase, CDK kinase,cyclin or a constitutively active Rb gene product, or an antisense RNA.

The present invention may also be described in certain broad aspects asa method of preventing restenosis in a subject following balloonangioplasty of either a coronary artery, renal artery, peripheral arteryor carotid artery, for example. In addition, the present invention maybe described in certain broad embodiments as a method of preventingrestenosis in a subject following balloon angioplasty of a vein as wouldbe used in a coronary artery bypass surgery, or other bioprostheticgrafts that might be used in the periphery. This method comprises thesteps of obtaining a viral vector comprising a cell cycle regulatorygene operatively linked to an SM22α promoter region dispersed in apharmaceutically acceptable solution and administering the solution tothe subject. The subject may be an animal subject and is preferably ahuman subject. In the practice of the method, the viral vector ispreferably a replication defective adenoviral vector and the gene maypreferably encode a constitutively active Rb gene product Rb, p53, cellcycle dependent kinase, CDK kinase, cyclin or a constitutively active Rbgene product.

An aspect of the invention is also a method of screening for identifyingsmooth muscle cell specific transcriptional control elements andparticularly those elements that work in trans. The method as providedherein preferably employs a reporter gene that confers on itsrecombinant hosts a readily detectable phenotype that is eitherexpressed or inhibited, as the case may be. Generally reporter genesencode (a) a polypeptide not otherwise produced by the host cell; or (b)a protein or factor produced by the host cell but at lower levels; or(c) a mutant form of a polypeptide produced by the host cell. Preferablythe gene encodes an enzyme which produces colorimetric or fluorometricchange in the host cell which is detectable by in situ analysis andwhich is a quantitative or semi-quantitative function of transcriptionalactivation. Exemplary enzymes include esterases, phosphatases, proteases(tissue plasminogen activator or urokinase) and other enzymes capable ofbeing detected by activity that generates a chromophore or a fluorophoreas will be known to those of skill in the art.

Examples of such a reporter gene are the E. coli β-galactosidase (β-gal)and firefly luciferase genes. The β-gal enzyme produces a color changeupon cleavage of the indigogenic substrate, indolyl-β-D-galactoside bycells expressing β-galactosidase. Thus, this enzyme facilitatesautomatic plate reader analysis of expression directly in microtiterwells containing transformants treated with candidate activators. Also,since the endogenous β-galactosidase activity in mammalian cellsordinarily is quite low, the analytic screening system usingβ-galactosidase is not hampered by host cell background. This enzymeoffers the further advantage that expression can be monitored in vivo bytissue analysis as described below.

Another class of reporter genes that confers detectable characteristicson a host cell are those that encode polypeptides, generally enzymes,that render their transformants resistant against toxins, e.g., the neogene, which protects host cells against toxic levels of the antibioticG418; a gene encoding dihydrofolate reductase, which confers resistanceto methotrexate, or the chloramphenicol acetyltransferase (CAT) gene:Other genes for use in the screening assay herein are those capable oftransforming hosts to express unique cell surface antigens, e.g. viralenv proteins such as HIV gp120 or herpes gD, which are readilydetectable by immunoassays.

In certain embodiments, the present invention may be described as arecombinant vector comprising an isolated SM22α promoter positionedadjacent a gene in a position to control expression of the gene. Thesplicing of nucleic acid sequences is well known in the art as describedabove and the insertion of such genes into vectors is also well known inthe art. The vector of the present invention may be a plasmid, aphagemid, an adenovirus or a retrovirus, for example. The type of vectordoes not in and of itself define the present invention, and therefore,any vector that can transfer genetic material into a cell to beexpressed in that cell will be useful in the present invention. It isalso understood that the nucleic acid segments may be transferred into acell by means such as liposomes, receptor ligand carriers, mechanicalmeans such as electroporation, etc. and that all such embodiments areencompassed within the claimed invention.

However, the recombinant vector of the present invention preferably is areplication deficient adenovirus or a high expression plasmid comprisingan SM22α promoter operatively joined to a gene, and wherein the gene isa cell cycle regulatory gene, such as Rb, p53, cell cycle dependentkinase, CDK kinase, cyclin or a constitutively active Rb.

It is understood that the method of inhibiting muscle contraction willhave utility in the treatment of palliation of a variety of diseasesthat arise from muscle cell contraction. Such diseases include, but arenot limited to Prinzinetal's angina, Raynaud's phenomenon, migraineheadache, a variety of collagen vascular diseases such as ELS,scleroderma, pulmonary hypertension, coronary arterial vasospasm, incontractile disorders of smooth muscle cells in the eye, gut, uterus,bladder, spleen, etc., or even in striated muscle spasms in paralysisvictims.

In a certain broad aspect the present invention may be described as amethod of promoting angiogenesis in a subject comprising the steps ofobtaining a nucleic acid segment comprising an angiogenesis factor geneoperatively linked to an SM22α promoter region; and transferring thenucleic acid segment into a smooth muscle cell to obtain a transfectedcell; wherein expression of the nucleic acid segment in the smoothmuscle cell promotes angiogenesis. In the practice of the method, thesmooth muscle cell may be a coronary arterial or venous smooth musclecell, or it may be a peripheral arterial or venous smooth muscle cell. Apreferred angiogenesis factor is VEGF for example. In certainembodiments of the method, the nucleic acid segment comprising anangiogenesis factor gene operatively linked to an SM22α promoter regionis contained in a viral or plasmid vector and the vector is administeredto a subject. In certain alternate embodiments, the transferring is doneex vivo and the method further comprises the steps of seeding abioprosthetic graft or stent with the transfected cells to obtain aseeded graft or stent; and placing the seeded graft or stent into acoronary or peripheral artery or vein of a subject.

The present invention may also be described in certain broad aspects asa method of inhabiting smooth muscle proliferation comprising the stepsof obtaining a nucleic acid segment comprising a cell cycle regulatorygene operatively linked to an SM22α promoter region; transferring thenucleic acid segment into a primary smooth muscle cell ex vivo to obtaina transfected cell; seeding a bioprosthetic graft or stent with thetransfected cell to obtain a seeded graft or stent; and placing theseeded graft or stent into a coronary or peripheral artery or vein of asubject, wherein expression of the cell cycle regulatory gene inhibitsproliferation of a smooth muscle cell.

Nucleic Acid Hybridization

The nucleic acid sequences disclosed herein will also find utility asprobes or primers in nucleic acid hybridization embodiments. As such, itis contemplated that oligonucleotide fragments corresponding to thesequence of SEQ ID NO:1 for stretches of between about 10 nucleotides toabout 20, for example, oligonucleotides of 11, 12, 13, 14, 15, 16, 17,18 or 19 nucleotides, or up to about 30 nucleotides inclusive of allsizes from 20 to 30 inclusive will find particular utility. It is alsounderstood that even longer sequences, e.g., up to 40, 50, 100, and evenup to full length, being more preferred for certain embodiments. Inaddition, the sequences, particularly intron sequences, disclosed hereinas SEQ ID NO:2 and SEQ ID NO:6 will find utility as probes and primersfor the discovery and isolation of related sequences. The ability ofsuch nucleic acid probes to specifically hybridize to SM22α genomicsequences will enable them to be of use in a variety of embodiments. Forexample, the probes can be used in a variety of assays for detecting thepresence of complementary sequences in a given sample. However, otheruses are envisioned, including the use of the sequence information forthe preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Nucleic acid molecules having stretches of 10, 20, 30, 50, or even of100 nucleotides or so, complementary to SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:6 will have utility as hybridization probes. These probes will beuseful in a variety of hybridization embodiments, such as Southern andnorthern blotting in connection with analyzing SM22α structural orregulatory genes in diverse cell lines and developmental stages and invarious species. The total size of fragment, as well as the size of thecomplementary stretch(es), will ultimately depend on the intended use orapplication of the particular nucleic acid segment. Smaller fragmentswill generally find use in hybridization embodiments, wherein the lengthof the complementary region may be varied, such as between about 10 andabout 100 nucleotides, or even up to 1419 or more according to thecomplementary sequences one wishes to detect.

The use of a hybridization probe of about 10, 15, or even 17 nucleotidesin length allows the formation of a duplex molecule that is both stableand selective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 15 to 20 nucleotides, or even longerwhere desired. For example, it is well established that a nucleotide of17 bases is sufficient to selectively hybridize to a target sequencecontained in a complex library such as a genomic library, for example,however smaller sequences are useful in less complex applications. Suchfragments may be readily prepared by, for example, directly synthesizingthe fragment by chemical means, by application of nucleic acidreproduction technology, such as the PCR™ technology of U.S. Pat. No.4,603,102 (herein incorporated by reference) or by introducing selectedsequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of SM22α genes or cDNAs. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by 0.02M-0.15M NaCl attemperatures of 50° C. to 70° C. Such conditions would be termed highstringency in that these conditions tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate SM22α-encodingsequences or promoters from related species, functional equivalents, orthe like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as 0.15M-0.9Msalt, at temperatures ranging from 20° C. to 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known which can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

The nucleic acid segments of the present invention, may be combined withother DNA sequences, such as polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, coding segments, andthe like, such that their overall length may vary considerably. It iscontemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol. Forexample, nucleic acid fragments may be prepared in accordance with thepresent invention which are up to 10,000 base pairs in length, withsegments of 5,000 or 3,000 being preferred and segments of about 1,000base pairs in length being particularly preferred.

It will be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of SEQ ID NO:1-SEQ IDNO:7. Therefore, DNA segments prepared in accordance with the presentinvention may also encode biologically functional equivalent promoters,proteins or peptides which have variant sequences, but essentially thesame function. Such sequences may arise as a consequence of codonredundancy and functional equivalency which are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.

In particular, one may wish to isolate the SM22α promoter from anotherspecies such as from a human. As such, one would obtain a human genomiclibrary from a commercial source, or one might create such a libraryfrom a human cell. One would then prepare a probe from the sequence ofSEQ ID NO:1, for example by labeling the nucleic acid with a radioactiveor fluorescent label. The genetic material from a series of clones wouldthen be hybridized to the probe and positive hybridizations would besubjected to a series of more stringent conditions until only amanageable number of positive clones remained to be tested by sequenceanalysis or by other means known in the art.

Alternatively, one might use a set of oligonucleotides based on thesequences disclosed herein as SEQ ID NO:1 to design primers to be usedin the PCR™ to amplify portions of an SM22α promoter, and the amplifiedDNA would then be used as a hybridization probe as above. The design anduse of PCR™ primers is well known in the art and would not require undueexperimentation in light of the present disclosure.

The GenBank accession number for the murine SM22α cDNA is L41154. TheGenBank accession number for the murine SM22α gene is L41161.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Identification and localization of transcriptional regulatoryelements that control SM22α gene expression. The data are from transienttransfection analyses of SM22α/luciferase reporter plasmids in thesmooth muscle cell line, A7r5. 15 mg of SM22α/luciferase reporterplasmid and 5 mg of the pMSVβgal reference plasmid were transientlytransfected into replicate cultures of A7r5 cells. Cells were harvested60 h after transfection, and cell extracts were assayed for bothluciferase and β-galactosidase activities. Luciferase activities (lightunits) were corrected for variations in transfection efficiencies asdetermined by β-galactosidase activities. Data are expressed asnormalized light units ±S.E.M. in the smooth muscle cell line, A7r5.

FIG. 1B. Transient transfection analyses of SM22α/luciferase reporterplasmids in primary rat aortic SMCs. Transient transfection analyseswere performed using a series of SM22α/luciferase reporter plasmids andprimary rat aortic SMCs as described in FIG. 1A. Data are expressed asnormalized light units ±S.E.M.

FIG. 2. Cellular-specificity of the 441-bp SM22α promoter. The p-441SM22luc (black bar) and pRSVL (hatched bar) plasmids were transientlytransfected into primary rat aortic SMCs (VSMC), A7r5, NIH 3T3 (3T3),COS-7, and Hep G2 cells and the normalized luciferase activities foreach respective plasmid was determined as described for FIG. 1A. Dataare expressed as normalized luciferase light units ±S.E.M.

FIG. 3A, FIG. 3B, FIG. 3C (Scanned images) and FIG. 3D. DNase Ifootprint analysis of the SM22α arterial SMC-specific promoter. (FIG.3A, FIG. 3B and FIG. 3C) Footprint analysis. Three overlapping genomicsubfragments (bp -441 to -256 (FIG. 3A), bp -256 to -89 (FIG. 3B), andbp -89 to +41 (FIG. 3C)) spanning the 482-bp (bp -441 to +42) SM22αpromoter were subjected to DNase I footprint analyses using nuclearextracts from the SMC line, A7r5 (which express high levels of SM22αmRNA) and NIH 3T3 cells. The sense (left panel) and antisense (rightpanel) strands of the three genomic subfragments were end-labeled andincubated in the absence (control) or presence of A7r5 and NIH 3T3 (3T3)of nuclear extracts before partial digestion with DNase I(concentrations varied from 5 U/ml to 22.5 U/ml). Standard Maxam andGilbert purine (G+A) sequencing reactions were run in parallel. The sixprotected regions identified on both strands with A7r5 nuclear extractswere designated smooth muscle elements (SME)-1-6, respectively, and arebracketed. DNase I hypersensitive sites are indicated with arrowheads.(FIG. 3D) Nucleotide sequence of the 441-bp SM22α arterial SMC-specificpromoter (SEQ ID NO:52=sense coding and SEQ ID NO:53=antisense coding).The six nuclear protein binding sites identified with A7r5 nuclearextracts are boxed. DNase I hypersensitive sites are indicated byarrowheads.

FIG. 4A and FIG. 4B (Scanned images). Electrophoretic mobility shiftassays (EMSAs) of the SME-1/CArG and SME-4/CArG nuclear protein bindingsites of the arterial SMC-specific SM22α promoter. (FIG. 4A):Identification of nuclear protein complexes that bind to SME-1.Radiolabeled oligonucleotides corresponding to the SME-1 binding sitewere subjected to EMSAs using 10 μg of nuclear extracts prepared fromprimary rat aortic SMCs (VSMCs). Some binding reactions included 5-50 ngof the indicated unlabeled competitor oligonucleotides or 1 μl of theindicated antiserum. Three specific complexes were detected and aredesignated A, B, and C, to the left of the autoradiogram. (FIG. 4B):Identification of nuclear protein complexes which bind to SME-4. EMSAswere performed using a radiolabeled SME-4 oligonucleotide probe asdescribed above. Four specific nuclear protein complexes were detectedand are designated A-D to the left of the autoradiogram.

FIG. 5A and FIG. 5B (Scanned images). EMSAs of the SME-5 (FIG. 5A) andSME-3 (FIG. 5B) nuclear protein binding sites of the arterialSMC-specific SM22α promoter. (FIG. 5A) EMSA performed with theradiolabeled SME-5 oligonucleotide probe and nuclear extracts preparedfrom primary rat aortic SMCs (VSMC), A7r5, WEHI (WE), and 70Z/3 (70Z)cells. Some binding reactions were pre-incubated with 1 μl of theindicated antiserum, or included the indicated unlabeled competitoroligonucleotides. Three nuclear protein complexes were identified andare designated A-C to the left of the autoradiogram. Complex A wasablated and supershifted (dashed arrow) by a-Sp1 antiserum. (FIG. 5B)EMSA performed with the radiolabeled SME-3 oligonucleotide probe andnuclear extracts prepared from primary rat aortic SMCs (VSMC), A7r5(A7)C3H10T1/2 (10T), NIH 3T3 (3T3), and EL4 cells. Some binding reactionsincluded between 10-75 ng of the indicated unlabeled competitoroligonucleotides. Three specific binding activities, designated A-C,were identified as denoted to the left of the autoradiogram. Nuclearprotein complexes that were ablated and supershifted by a-YY1-specificantisera are indicated with arrows to the left of the EMSA. Of note,complex C was present only in nuclear extracts prepared from SMClineages (arrow). In addition, three unique nuclear protein complexeswere present in non-SMC nuclear extracts but not in SMC extracts (dashedarrows).

FIG. 6 (Scanned image). EMSA of the SME-6/CRE nuclear protein bindingsite of the arterial SMC-specific SM22α promoter. (A) EMSA performedwith the radiolabeled SME-6 oligonucleotide probe and nuclear extractsprepared from primary rat aortic SMCs (VSMC), A7r5 (A7), C2C12 myotubes(C2T), C3H10T1/2 (10T), NIH 3T3 (3T3) and EL4 cells. Some bindingreactions included the indicated unlabeled competitor oligonucleotidesor the indicated antiserum. Four specific nuclear protein complexes,designated A-D, are denoted to the left of the autoradiogram. Thecomplexes that were ablated and/or supershifted by α-CREB-1, α-ATF-1,α-Sp1 and α-YY1 antiserum are shown to the left of the autoradiogram.

FIG. 7. Schematic representation of the cis-acting elements and thetrans-acting factors identified by DNase I footprint and EMSAs analysesthat bind to the SM22α promoter. Six nuclear protein binding sites wereidentified by DNase I footprint analysis in the 441-bp arterialSMC-specific SM22α promoter that were designated SME-1-6, respectively.Trans-acting factors identified by EMSA that bind to each nuclearprotein binding site are shown above or below each cis-acting element.Binding sites for SRF and ternary complex factors (T) (SME-1 and SME-4),Sp1 (SME-1, -2, -5, -6), YY1 (SME-3, -4, -6), CREB-1 (SME-6) and ATF-1(SME-6) were identified. Of note, nuclear protein complexes that couldnot be positively identified by antibody supershift reactions are shownin gray below the nuclear protein binding site to which they bind.

FIG. 8A and FIG. 8B. Functional analysis of the cis-acting elements thatcontrol transcription of the SM22α promoter in arterial SMCs. Mutationswere introduced into the 441-bp SM22α promoter as described below. Themutant promoters were subcloned into the pGL2-Basic luciferase reporterplasmid, and the resulting plasmids were transfected into primary rataortic SMCs. A schematic representation of the SM22α promoter and themutated cis-acting elements (indicated by black) are shown to the leftof the graph. Luciferase activities, corrected for differences intransfection efficiencies, are shown as a percent of the luciferaseactivity observed with the p-441SM22luc plasmid ±S.E.M. Eachtransfection was repeated at least three times. (FIG. 8A) Effects ofmutation of the SME-1/CArG and SME-4/CArG nuclear protein binding siteson SM22α promoter activity in arterial SMCs. (FIG. 8B) Effect ofmutation of the SME-2, -3, -5, and -6 sites on SM22α promoter activityin arterial SMCs.

FIG. 9. Analysis of 5' deletions from the SM22α promoter as measured bynormalized luciferase activity. Deletion of bp -445 through -400 (column-399) reduces luciferase activity by approximately 50% compared to thecomplete SM22α promoter (column -445). Further deletions of bp -445through -301 (columns -352 and -300) does not further reduce luciferaseactivity. Deletion of bp -445 through -163 (columns -252, -199 and -162)nearly eliminated all measurable SM22α activity.

FIG. 10 (Scanned image). EMSA analysis of DNA region encompassing acis-acting, positive transcriptional regulatory element. A radiolabeleddouble-stranded oligonucleotide corresponding to bp -445 to -389specifically binds nuclear proteins extracted from either A7r5 SMCs orcultured rat tracheal SMCs (band B-1).

FIG. 11. Sequence analysis of bp -445 to -400, SEQ ID NO:20, of theSM22α promoter (the sequences depicted in this figure are SEQ ID NO:54and SEQ ID NO:55)reveals the presence of 2 potential zeste-binding siteswith 5' ends at bp -435 and -421, respectively. Methylation interferenceassays revealed interference with A7r5 nuclear protein binding atseveral bases within the 20-bp span from -435 to -416. Protected basesare in bold print. Underlined bases indicate nucleotides that weremutated to determine their effect on nuclear protein binding. A-sitedesignates the portion of the intervening sequence between thezeste-sites (Z1 and Z2) that was also mutated to determine its effect onnuclear protein binding.

FIG. 12. Transient transfection assays demonstrate that mutation of theZ1 zeste-binding site of the 441-bp SM22α promoter reducestranscriptional activity to approximately one-half of the wild type441-bp SM22α promoter in A7r5 SMCs (as assessed by normalized luciferaseactivity), confirming the importance of the Z1 site as an importantpositive transcriptional regulatory sequence within the SM22α promoter.

FIG. 13. Normalized luciferase activity of recombinant vectorscontaining the 482-bp (p-441SM22luc), the 341-bp (p-300SM22luc) or the201-bp (p-162SM22luc) SM22αpromoter and transfected into cultured rattracheal SMCs compared to the luciferase activity of the highly activeLTR promoter, of the RSV virus, present in the recombinant vector pRSVL.Activity of the 482-bp SM22α promoter is equivalent to the activity ofthe LTR promoter.

FIG. 14. (Scanned image). Ribonuclease protection assay (RPA) detectsthe presence of protected fragments of 167 and 78 nucleotides in RNAfrom cultured rat tracheal SMCs transfected with p-441SM22luc andp-441SM22-8pep, respectively, indicating that luciferase-encoding mRNAand MIRICRKK, SEQ ID NO:19,-encoding MRNA is present and the promoter isactive.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention arises from the isolation and characterization ofa smooth muscle cell specific promoter region that, in its naturallyoccurring state, controls the expression of the SM22α gene. Theinventors have shown that this isolated promoter region may beoperatively joined to a heterologous structural gene and will controlthe expression of that gene specifically in smooth muscle cells andother myogenic cell lineages including an embryonic skeletal musclecell. An important element of the present invention is that, unlikeother known smooth muscle cell promoters, the SM22α promoter is cellcycle independent and is thus not down-regulated when the cell entersthe proliferative state. The promoter sequence of the present inventionwill be useful in the expression of heterologous genes in a smoothmuscle cell, in the discovery of trans and cis acting transcriptionalcontrol elements that affect smooth muscle cell gene expression and as aprobe to isolate SM22α genes and promoters. In particular, the presentinvention will find use in the prevention of restenosis followingballoon angioplasty or other arterial injury, in the promotion ofangiogenesis in graft or stent implants and in the treatment orprevention of asthma among other smooth muscle cell proliferativediseases.

SM22α is expressed exclusively in smooth muscle-containing tissues ofadult animals and is one of the earliest markers of differentiatedsmooth muscle cells (SMCs). SM22α is a 6.2 kb single copy gene composedof five exons. SM22α mRNA is expressed at high levels in the aorta,uterus, lung, and intestine, and in primary cultures of rat aortic SMCs,and the SMC line, A7r5. In contrast to genes encoding SMC contractileproteins, SM22α gene expression is not decreased in proliferating SMCs.Transient transfection experiments demonstrated that 441-bp of SM22α 5'flanking sequence was necessary and sufficient to program high leveltranscription of a luciferase reporter gene in both primary rat aorticSMCs and A7r5 cells. DNA sequence analyses revealed that the 441-bppromoter contains two CArG/SRF boxes, a CACC box, and one potentialMEF-2 binding site, cis-acting elements which are each importantregulators of striated muscle transcription. Taken together, thesestudies have identified the murine SM22α promoter as an excellent modelsystem for studies of developmentally regulated, lineage-specific geneexpression in SMCs.

As disclosed herein, the murine SM22α cDNA and gene have been isolatedand structurally characterized. Using the murine SM22α cDNA as amolecular probe, the tissue distribution and cell cycle-regulatedpattern of SM22α gene expression have been defined. In addition, it hasbeen demonstrated that the immediate 5' flanking region of the SM22αgene is necessary and sufficient to direct high-level,lineage-restricted expression of the SM22α gene in both primary vascularSMCs and the SMC line, A7r5. Finally, it has been demonstrated that theminimal SM22α promoter lacks a binding site for the bHLH family ofmyogenic transcription factors. These data are relevant to understandingthe underlying transcriptional program that regulates SMCdifferentiation.

The unique contractile properties of SMCs and their ability toreversibly modulate their phenotype from primarily contractile toprimarily synthetic, distinguishes this myogenic lineage from both theskeletal and cardiac muscle cell lineages. However, in contrast to thestriated muscle lineages (for review see Olson, 1990; Tapscott et al.,1991; Olson, 1993; Olson et al., 1994), relatively little is currentlyunderstood about the cis-acting sequences and trans-acting factors thatregulate gene expression in SMCs, due, in part, to the poorly understoodlineage relationships of SMCs, which appear to develop from multiplelocations throughout the embryo, as well as to the relative paucity ofSMC-specific markers (Gonzalez-Crussi, 1971; Lelievre et al., 1975;Murphy et al., 1978; Hirakow et al., 1981; Pardanaud et al., 1989; Pooleet al., 1989; Hood et al., 1992). The data disclosed herein demonstratethat the level of SM22α protein expression is regulated at the level ofgene expression. However, in contrast to the smooth muscle myosin heavychain, and possibly the γ-enteric actin gene, which are expressedexclusively in SMCs (Rovner et al., 1986; Sawtell et al. 1989; Aikawa etal., 1993; Frid et al., 1993; Miano et al., 1994), SM22α is expressed inother myogenic cell lineages including the embryonic skeletal musclecell lineage C2C12. In this regard, it is noteworthy that the SM22α geneis expressed in undifferentiated skeletal myoblasts, which do notexpress myofibrillar protein isoforms, and that SM22α gene expression isnot down-regulated in conjunction with other SMC contractile proteinsduring serum-induced SMC proliferation. Taken together, these datasuggest, that the SM22α gene is not regulated in a coordinated mannerwith other smooth muscle contractile proteins. Therefore, the isolatedSM22α promoter of the present invention contains cis-acting sequencesthat regulate SM22α gene expression in SMCs and serves as a valuable andunique tool for targeting gene expression to both contractile/arrestedand synthetic/proliferative SMCs in the arterial wall in vivo.

This differential pattern of SM22α gene expression in several myogeniclineages suggests that distinct transcriptional programs have evolved topermit the regulated expression of a single gene in multiple celllineages. However, it is noteworthy that Olson and coworkers (Lilly etal., 1995) recently reported that a null mutation of the MADS boxtranscription factor D-MEF2 gene in Drosophila resulted in failure ofsomatic, cardiac and visceral muscles to differentiate. These datasuggest that this evolutionarily conserved family of transcriptionfactors may play a critical role in coordinating muscle differentiationacross lineages. Thus, it will be of interest to determine thefunctional role of the A/T-rich potential MEF-2/rSRF (8/10 bp sequenceidentity) binding site located within the minimal murine SM22α promoter.In this respect, the SM22α promoter may serve as a useful target withwhich to dissect the functional role of the four individual MEF-2/rSRFfamily members expressed in vertebrate species (versus the singleD-MEF-2 gene in Drosophila) in the smooth muscle lineage. Similarly, twoconsensus CArG box/SRF binding sites were identified in the minimalSM22α promoter. This motif, which has been identified in multipleskeletal and cardiac-specific transcriptional regulatory elements(Gustafson et al., 1988), is also present in the smooth muscle α-actinpromoter (Carroll et al., 1988; Min et al., 1990; Blank et al., 1992)suggesting that it may play a role in the coordinate regulation of genesexpressed in SMCs. Finally, a consensus CACC box was identified in theminimal SM22α promoter. This nuclear protein binding site is present inmultiple skeletal and cardiac-specific transcriptional regulatoryelements, where it has been demonstrated to function in conjunction withother lineage-specific nuclear protein binding sites (Parmacek et al.,1990; Parmacek et al., 1994; Jaynes et al., 1988; Devlin et al., 1989;Edmondson et al., 1992).

Another family of transcription factors that have been implicated in SMCdevelopment are the homeodomain proteins. In vertebrate species,homeobox proteins are generally involved in morphogenesis andestablishment of body plan (reviewed in (Carroll, 1995; Shashikant etal., 1991). The mesoderm-specific homeodomain protein, MHox, which ismost closely related to the paired family of homeodomain proteins, isfirst expressed during murine development in mesodermal cells within thelateral plate mesoderm and visceral arches beginning at day 8.5.Subsequently, it is expressed abundantly in the uterus, heart andskeletal muscle leading to the suggestion that MHox may play a role inSMC (and striated muscle) pattern formation (Cserjesi et al., 1992).However, mice carrying a null mutation in the MHox gene only exhibitdefects in skeletal organogenesis resulting from a defect in theformation and growth of chondrogenic and osteogenic precursors (Martinet al., 1995). The finding that only a subset of embryonic structuresderived from MHox-expressing cells is affected by this mutation has ledto the suggestion that other homeobox proteins expressed in SMCs canfunctionally substitute for MHox (Gottesdiener et al., 1988). Severalpotential candidates have been suggested including the closely-relatedhomeobox protein S8 and the more distantly-related homeobox protein Gax,both of which are expressed abundantly in mesodermal derivatives (Gorskiet al., 1993; Martin et al., 1995). Finally, it has been suggested thatprotein-protein interactions between homeodomain proteins and MADS boxtranscription factors (see above) may modulate the transcriptionalactivity of the MADS box proteins (Grueneberg et al., 1992). Forexample, the human MHox homologue Phox1, enhances the DNA-bindingactivity of SRF in vitro and functionally synergizes with SRF in vivo,suggesting that MHox (or a functionally-related homeodomain protein)could establish cell identity, in part, by determining which genes areactivated in response to generic inductive signals which are transducedby ubiquitously-expressed transcription factors such as SRF (Grueneberget al., 1992).

Current developmental paradigms suggest that tissue-specific geneexpression is ultimately regulated by the expression of lineage-specificor lineage-restricted transcription factors (Olson, 1990; Tapscott etal., 1991; Olson, 1993; Olson et al., 1994). Interestingly, sequenceanalyses of the minimal SM22α promoter failed to reveal a consensus bHLHmyogenic transcription factor/E-box binding site. Consistent with thisobservation, myogenic bHLH family members, including MyoD, myogenin,myf-5 and MRF-4/herculin/myf-6, are not expressed in SMCs and nullmutations of the MyoD, myogenin and myf-5 genes, respectively, had noeffect on smooth muscle cell specification or differentiation in vivo(Hasty et al., 1993; Rudnicki et al., 1993). Similarly, the minimalSM22α promoter lacked a consensus binding site for GATA-4, atranscription factor that has been demonstrated to transactivatemultiple cardiac-specific transcriptional regulatory elements innon-muscle cell lines (Ip et al., 1994; Grepin et al., 1994). Takentogether, these studies suggest that potentially novel SMC-specifictranscription factors may play a key role in regulating SMC-specifictranscription. Future studies utilizing the SM22α promoter as a modelsystem should provide fundamental insight into the molecular mechanismsthat regulate SMC-specific transcription and differentiation.

In one aspect, the present invention provides a process of directing andregulating gene expression in a smooth muscle cell. In accordance withthat process, a gene operatively joined to an SM22α promoter isdelivered to a smooth muscle cell and the smooth muscle cell is thenmaintained under physiological conditions and for a period of timesufficient for the gene to enter the smooth muscle cell, for the gene tobe transcribed and in certain embodiments, for the product of that geneto be expressed. Delivery is preferably by transfection with an aplasmid or a high expression plasmid, adenovirus, p21 virus, raussarcoma virus, or other virus vector construct capable of transfecting asmooth muscle cell, and comprising an SM22α promoter operatively joinedto a coding sequence that encodes the gene product.

The use of adenovirus as a vector for cell transfection is well known inthe art. Adenovirus vector-mediated cell transfection has been reportedfor various cells (Stratford-Perricaudet, et al., 1992). An adenovirusvector of the present invention is replication defective. A virus isrendered replication defective by deletion of the viral early region 1(E1) region. An adenovirus lacking an E1 region is competent toreplicate only in cells, such as human 293 cells, which expressadenovirus early region 1 genes from their cellular genome. Thus, suchan adenovirus cannot replicate in cells that do not provide the earlygene product of the E1 region. In a preferred embodiment, an adenovirusvector used in the present invention is lacking both the E1 and the E3early gene regions. Thus, it is most convenient to introduce the codingsequence for a gene product at the position from which the E1 and/or E3coding sequences have been removed (Karlsson et al., 1986). Preferably,the E1 region of adenovirus is replaced by the coding DNA sequence orgene. However, the position of insertion within the adenovirus sequencesis not critical to the present invention. Techniques for preparing suchreplication defective adenoviruses are well known in the art asexemplified by Ghosh-Choudhury et al., McGrory et al., 1988, and Gluzmanet al., 1982.

A wide variety of adenovirus vectors can be used in the practice of thepresent invention. An adenovirus vector can be of any of the 42different known serotypes of subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material for production of areplication-defective adenovirus vector. Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

In order to replicate the virus, the vector is co-transfected into 293cells together with a plasmid carrying the complete adenovirus type 5genome. Preferred plasmids may also confer ampicillin and tetracyclineresistance due to insertion of the appropriate sequences into the virusgenome. The molecular strategy employed to produce recombinantadenovirus is based upon the fact that, due to the packaging limit ofadenovirus, the plasmid cannot efficiently form plaques on its own.Therefore, homologous recombination between the desired construct andthe co-transfected plasmid within a transfected cell results in a viablevirus that can be packaged and form plaques only on 293 cells.

Co-transfection is performed in accordance with standard procedures wellknown in the art. By way of example, 293 cells are cultured inDulbecco's modified Eagle's medium containing 10% fetal calf serum in ahumidified 5% CO₂ atmosphere. Confluent 10 cm dishes are split intothree 6 cm dishes. On the following day, the cells are cotransfected incalcium phosphate with HeLa DNA as carrier. Six hours after addition ofthe DNA to the cells, a 15% glycerol stock is used to boost transfectionefficiency and the cells are overlaid with 0.65% Noble agar in DMEMcontaining 2% FCS, 50 mg/ml penicillin G, 10 mg/ml streptomycin sulfate,and 0.25 mg/ml fingizone (GIBCO, Grand Island, N.Y.). Monolayers areincubated for approximately 10 days until the appearance of viralplaques.

These plaques are picked, suspended in DMEM containing 2% FCS, and usedto infect a new monolayer of 293 cells. When greater than 90% of thecells show infection, viral lysates are subjected to a freeze/thaw cycleand designated as primary stocks. Recombinant virus with the correctstructure is verified by preparation of viral DNA fromproductively-infected 293 cells, restriction analysis, and Southernblotting. Secondary stocks are subsequently generated by infecting 293cells with primary virus stock at a multiplicity of infection of 0.01and incubation until lysis.

The particular cell line used to propagate the recombinant adenovirusesof the present invention is not critical to the present invention.Recombinant adenovirus vectors can be propagated on, e.g., human 293cells, or in other cell lines that are permissive for conditionalreplication-defective adenovirus infection, e.g., those which expressadenovirus E1 gene products "in trans" so as to complement the defect ina conditional replication-defective vector. Further, the cells can bepropagated either on plastic dishes or in suspension culture, in orderto obtain virus stocks thereof.

When the vector is to be delivered to an animal subject, a preferredmethod is to percutaneously infuse an adenovirus vector construct into ablood vessel that perfuses smooth muscle cells (WO 9411506, Barr et al.,1994, both incorporated herein by reference) by intravenous orintra-arterial injection. Methods of delivery of foreign DNA are knownin the art, such as containing the DNA in a liposome and infusing thepreparation into an artery (LeClerc et al., 1992, incorporated herein byreference), transthoracic injection (Gal et al., 1993, incorporatedherein by reference). Other methods of delivery may include coating aballoon catheter with polymers impregnated with the foreign DNA andinflating the balloon in the region of arteriosclerosis, thus combiningballoon angioplasty and gene therapy (Nabel et al., 1994, incorporatedherein by reference).

After delivery of an adenovirus vector construct to a smooth musclecell, that cell is maintained under physiological conditions and for aperiod of time sufficient for the adenovirus vector construct to infectthe cardiac cell and for cellular expression of a coding sequencecontained in that construct. Physiological conditions are thosenecessary for viability of the SMOOTH muscle cell and include conditionsof temperature, pH, osmolality and the like. In a preferred embodiment,temperature is from about 20° C. to about 50° C., more preferably fromabout 30° C. to about 40° C. and, even more preferably about 37° C. pHis preferably from a value of about 6.0 to a value of about 8.0, morepreferably from about a value of about 6.8 to a value of about 7.8 and,most preferably about 7.4. Osmolality is preferably from about 200milliosmols per liter (mosm/L) to about 400 mosm/l and, more preferablyfrom about 290 mosm/L to about 310 mosm/L. Other physiologicalconditions needed to sustain smooth muscle cell viability are well knownin the art.

It should also be pointed out that because the adenovirus vectoremployed is replication defective, it is not capable of replicating inthe cells that are ultimately infected. Moreover, it has been found thatthe genomic integration frequency of adenovirus is usually fairly low,typically on the order of about 1%. Thus, where continued treatment isrequired, it may be necessary to reintroduce the virus every 6 months toa year. In these circumstances, it may therefore be necessary to conductlong term therapy, where expression levels are monitored at selectedintervals.

An adenovirus vector construct is typically delivered in the form of apharmacological composition that comprises a physiologically acceptablecarrier and the adenovirus vector. An effective expression-inducingamount of such a composition is delivered. As used herein, the term"effective expression-inducing amount" means that number of virus vectorparticles necessary to effectuate expression of a gene product encodedby a coding sequence contained in that vector. Means for determining aneffective expression-inducing amount of an adenovirus vector constructare well known in the art. An effective expression-inducing amount istypically from about 10⁷ plaque forming units (pfu) to about 10¹⁵ pfu,preferably from about 10⁸ pfu to about 10¹⁴ pfu and, more preferably,from about 10⁹ to about 10¹² pfu.

As is well known in the art, a specific dose level for any particularsubject depends upon a variety of factors including the infectivity ofthe adenovirus vector, the age, body weight, general health, sex, diet,time of administration, route of administration, rate of excretion, andthe severity of the particular disease undergoing therapy.

In that adenovirus is a virus that infects humans, there may be certainindividuals that have developed antibodies to certain adenovirusproteins. In these circumstances, it is possible that such individualsmight develop an immunological reaction to the virus. Thus, where animmunological reaction is believed to be a possibility, one may desireto first test the subject to determine the existence of antibodies. Sucha test could be performed in a variety of accepted manners, for example,through a simple skin test or through a test of the circulating bloodlevels of adenovirus-neutralizing antibodies. In fact, under suchcircumstances, one may desire to introduce a test dose of on the orderof 1×10⁵ to 1×10⁶ or so virus particles. Then, if no untoward reactionis seen, the dose may be elevated over a period of time until thedesired dosage is reached, such as through the administration ofincremental dosages of approximately an order of magnitude.

In another aspect, the present invention relates to pharmaceuticalcompositions that may comprise an adenovirus vector gene constructdispersed in a physiologically acceptable solution or buffer. Acomposition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, wellknown, nontoxic, physiologically acceptable carriers, adjuvants, andvehicles as desired. The term parenteral as used herein includesintravenous, intramuscular, intraarterial injection, or infusiontechniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol. Among the acceptable vehiclesand solvents that may be employed are water, Ringer's solution, andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one purifies thevector sufficiently to render it essentially free of undesirablecontaminant, such as defective interfering adenovirus particles orendotoxins and other pyrogens such that it will not cause any untowardreactions in the individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. The following techniques and materials were used in thepractice of the examples unless otherwise indicated.

Isolation of Murine SM22α cDNA Clones

The coding region of the murine SM22α cDNA was isolated by performinglow stringency PCRTM using murine uterine RNA and synthetic 5' and 3'oligonucleotide PCR™ primers constructed from the previously publishedsequence of the rat SM22α cDNA (Nishida et al., 1993). The 5' PCR™primer was constructed to be identical to the first 34-bp of the ratSM22α cDNA with the addition of a 5' EcoRI site (5'ATCGAATTCCGCTACTCTCCTTCCAGCCC ACAAACGACCAAGC 3', SEQ ID NO:10). The 3'primer was constructed to include the reverse complement of bp 759 to782 of the rat SM22α cDNA with an additional 3' HindIII restriction site(5' ATCAAGCTTGGTGGGAGCTGCCCATGTGCAGTC 3', SEQ ID NO:11). PCR™ reactionproducts were subcloned into EcoRI/HindIII-digested pGEM7Z (Promega,Madison, Wis.) as described elsewhere (Parmacek et al., 1989). Thenucleotide sequence of the murine SM22α cDNA was confirmed by sequencingof the full-length murine SM22α genomic clone. MacVector DNA sequencingsoftware (Kodak/IBI, Rochester, N.Y.) was used for DNA sequenceanalyses.

To isolate the 3' untranslated region of the SM22α cDNA, 5×10⁵recombinant clones from an oligo-(dT) primed λgt11 C2C12 myotube cDNAlibrary were screened with the [³² P]-labeled murine SM22α cDNA probe(bp 29-811) as described previously (Parmacek et al., 1992). Twelveclones were purified to homogeneity and analyzed by Southern blotanalyses as described (Parmacek et al., 1992). Two independent clones,each of which contained a poly(A) tail, were subcloned intoEcoRI-digested pGEM7Z and their nucleotide sequences determined. Thenucleotide sequence of the 5'-untranslated region was determined fromthe sequence of the SM22α genomic clone. The 5'-untranslated region waslocalized on the genomic clone by Southern blot hybridizations, inaddition to RNase protection and primer extension analyses as describedbelow.

Isolation of Murine SM22α Genomic Clones

Approximately 1×10⁶ recombinant phage from a murine 129SV Lambda FIX IIgenomic library (Stratagene, La Jolla, Calif.) were screened with the783-bp murine SM22α cDNA probe (bp 29-811) labeled with [α-³² P]dCTP,and three positive clones were purified to homogeneity as describedpreviously (Parmacek et al., 1992). One clone (SM22-13a) was found toinclude the entire coding region of the SM22α gene and 9-kb of 5'flanking sequence and was used for all subsequent subcloning andsequencing experiments.

Southern Blot Analyses

High molecular weight DNA was prepared from the tails of strain 129SVmice as described previously (Parmacek et al., 1989). Southern blottingand hybridization to the radiolabeled 783-bp murine SM22α cDNA probewere performed as described previously (Parmacek et al., 1989). Lowstringency washing conditions were 2×SSC, 0.1% SDS at 50° C. Highstringency washing conditions were 0.1×SSC, 0.1% SDS at 68° C.

Northern Blot Analyses

Tissues were isolated from 12-week old 129SV mice (Jackson Laboratories)as described previously (Parmacek et al., 1989). Animals were housed andcared for according to NIH guidelines in the University of ChicagoLaboratory Animal Medicine Veterinary Facility. RNA was prepared fromorgan samples and from cultures of primary rat aortic SMCs, the rat SMCline A7r5, and non-smooth muscle cell lines including murine NIH 3T3cells, murine C3H10T1/2 cells, monkey COS-7 cells, murine C2C12myoblasts and myotubes, human HepG2 cells, and murine EL-4 cells by thesingle step guanidinium isothiocyanate protocol (Chomczynski, 1993).Northern blotting was performed using 10 mg of RNA per sample asdescribed previously with the exception that 36 mg/ml of ethidiumbromide was added to the RNA resuspension buffer in order to permitquantitation of the 28S and 18S ribosomal RNA subunits in each lane.Probes included the 783-bp (bp 29-811) murine SM22α cDNA and the 754-bp(bp 659-1404) murine calponin cDNA probe. Quantitative image analyseswere performed using a Molecular Dynamics Phosphorlmager (Sunnyvale,Calif.).

Primer Extension, 5' RACE, and RNase Protection Analyses

A 25-mer oligonucleotide probe constructed to include the reversecomplement of base pairs +80 to +104 of the SM22α gene (5'TGCCGTAGGATGGACCCTTGTTGGC 3', SEQ ID NO:12) was 5' end labeled with[γ-³² P]ATP and T4 polynucleotide kinase. 40 mg of mouse uterine RNA washybridized to 2×10⁶ DPM of labeled probe and primer extension reactionsperformed at 42° C., 50° C. and 56° C. as described previously (Parmaceket al., 1992). 5' RACE was performed using murine uterine RNA and asynthetic antisense cDNA probe corresponding to bp 234 to 258 of themurine SM22α cDNA according to the manufacturer's instructions (PerkinElmer, Norwalk, Conn.). RNase protection analyses were performed bysubcloning the -441 to +41 murine SM22α genomic subfragment including asynthetic 3' HindIII linker into PstI/HindIII-digested pGEM4Z andperforming in vitro transcription of the antisense strand of the genomicsubfragment with T7 polymerase of the NcoI-linearized plasmid (NcoI cutsat bp -88 of the genomic clone) in order to obtain an antisense cRNAprobe corresponding to bp -88 to +44. Of note, the HindIII linker sharessequence identity with the SM22α cDNA resulting in a cRNA probe withsequence identity initiated at bp +44 (not +41) of the SM22α genomicclone. The 142-bp probe was labeled with [α-³² P]UTP and RNaseProtection Analyses were performed using the RPAII™ kit (Ambion, Austin,Tex.) according to the manufacturer's instructions. Antisense cRNA proberadiolabeled by incorporation of α-[³² P]-UTP is synthesized by in vitrotranscription from linearized pBluescriptIIKST7-lacZ, which contains thelacZ gene upstream of the T7 RNA polymerase promoter, using theMaxiScript™ kit (Ambion, Austin, Tex.). Band intensity is quantified byPhosphorlmager™, as previously for southern analyses described above.

Cell Culture

The rat cell line A7r5 which was derived from embryonic thoracic aortawas grown in Dulbecco's Modified Essential Media (GIBCO) supplementedwith 10% fetal bovine serum (GIBCO) and 1% penicillin/streptomycin. Thehuman hepatocellular carcinoma cell line Hep G2 was grown in ModifiedEagle's Medium supplemented with 10% fetal bovine serum and 0.1 mM MEMnon-essential amino acids (GIBCO). Murine lymphoma-derived EL4 cellswere grown in Dulbecco's modified Eagle's Media supplemented with 10%horse serum (GIBCO). Murine NIH 3T3 cells, C3H10T1/2 cells, C2C12myoblasts and myotubes were grown as described previously (Parmacek etal., 1990; Parmacek et al., 1994). Primary cultures of rat aortic SMCswere isolated from 12-16 week old Sprague Dawley rats (Charles RiverLaboratories, Wilmington, Mass.) using the method described previously(Chang et al., 1995). Virtually all cells isolated using this methodstain positive with anti-smooth muscle actin monoclonal antiserum. Inall studies, only early passage (passage 2 or 3) rat aortic SMCs wereutilized. For the cell cycle analyses, SMCs from the third passage wereplaced in serum-free medium (50% Dulbecco's minimal essential medium(DMEM), 50% Ham's F-12, L-glutamine (292 mg/ml), insulin (5 mg/ml),transferrin (5 mg/ml), selenious acid (5 ng/ml)) for 72 hrs in order tosynchronize the cells in G₀ /G₁ as described previously (Chang et al.,1995). Following 72 hrs of serum starvation, cells were stimulated toproliferate by incubation in medium containing 45% DMEM, 45% Ham's F-12and 10% FBS. Mouse WEHI B-cells and mouse 70Z/3 pre-B lymphocytes weregrown as described previously (Morrisey et al., 1996).

DNase I Footprinting

Nuclear extracts were prepared from the smooth muscle cell line, A7r5(which express high levels of SM22α mRNA (Solway et al., 1995)) and NIH3T3 cells as described previously (Parmacek et al., 1992). Threeoverlapping genomic subfragments (bp -441 to -256, bp -256 to -89, andbp -89 to +41) spanning the 482-bp SM22α promoter were analyzed. DNase Ifootprint analyses were performed with 100-150 mg of nuclear extractsprepared from the smooth muscle cell line, A7r5, or NIH 3T3 fibroblastsand the end-labeled sense and antisense strands of the murine SM22αpromoter as described previously (Parmacek et al., 1994). Standard Maxamand Gilbert (G+A) sequencing reactions were run in parallel to identifythe protected sequences.

Electrophoretic Mobility Shift Assays (EMSAs)

Nuclear extracts were prepared from low passage number primary rataortic SMCs, A7r5 cells, NIH 3T3 cells, C3H10T1/2 cells, C2C12 myotubes,WEHI, 70Z/3 and EL4 cells as described by Dignam et al. (Dignam et al.,1983). EMSAs were performed in 0.25×TBE (1×TBE is 100 mM Tris, 100 mMboric acid and 2 mM EDTA) as described previously (Ip et al., 1994). Thefollowing complementary oligonucleotides (corresponding to each nuclearprotein binding site identified by DNaseI footprint analysis or nuclearprotein binding sites containing the specific mutations indicated(mutated nucleotides are underlined)) were synthesized with BamHI andBglII overhanging ends:

SME-1-5' AAGGAAGGGTTTCAGGGTCCTGCCCATAAAAGGTTTTTCCCGGCCGC 3' (SEQ IDNO:21);

μSME-1-5' AAGGAAGGGTTTCAGGGTCCTGCCCATAGATCTTTTTTCCCGGCCGC 3' (SEQ IDNO:22);

SME-2-5' CCGCCCTCAGCACCGCCCCGCCCCGAGGCCCGCAGCATGTCCG 3' (SEQ ID NO:23);

μSME-2-5' CCGCCCTCAGCACCGCGGATCCCCGACCCCCGCAGCATCTCCG 3' (SEQ ID NO:24);

SME-3-5' CTCCAAAGCATGCAGAGAATGTCTCCGGCTGCCCCCG 3' (SEQ ID NO:25);

μSME-3-5' CTCGGATCCATGCTAGCAATGAATTCGGCTGCCCCCG 3' (SEQ ID NO:26);

SME-4-5' TCCAACTTGGTGTCTTTCCCCAAATATGGAGCCTGTGTGGAGTG 3' (SEQ ID NO:27);

μSME-4-5' TCCAACTTGGTGTCTTTCCCCAAGGATCCAGCCTGTGTGGAGTG 3+ (SEQ IDNO:28);

μSRF/SME-4-5' TCCAACTTGGTGTCTTTCCCCGGATATGGAGCCTGTGTGGAGTG 3' (SEQ IDNO:29);

μYY1/SME-4-5' TCCAACTTGGTGTCTTTCCCCAAATTAGGAGCCTGTGTGGAGTG 3' (SEQ IDNO:30);

SME-5-5' GGGCAGGGAGGGGCGCCAGCG 3' (SEQ ID NO:31);

μSME-5-5' GGGCAGGTACCGAATTCAGCG 3' (SEQ ID NO:32);

SME-6-5' GGACGGCAGAGGGGTGACATCACTGCCTAGGCGGCCG 3' (SEQ ID NO:33);

μCREB/SME-6-5' GGACGGCAGAGGGGATCCATGCTGCCTAGGCGGCCG 3' (SEQ ID

NO:34);

μYY1I/SME-6-5' GGACGGCAGAGGGGATCCATCACTGCCTAGGCGGCCG 3' (SEQ ID

NO:35);

Sp1-5' CTGGCTAAAGGGGCGGGGCTTGGCCAGCC 3' (SEQ ID NO:36);

CREB/TCRα-5' CTCCCATTTCCATGACGTCATGGTTA 3' (SEQ ID NO:37).

For cold competition studies, 5 to 100 ng of unlabeled competitoroligonucleotide was included in the binding reaction mixture. Forantibody supershift studies, 1 μl of either rabbit preimmune, affinitypurified rabbit or mouse IgG (Santa Cruz), α-SRF rabbit polyclonalantiserum (Santa Cruz, sc-335X), α-Sp1 rabbit polyclonal IgG (SantaCruz, sc-059X), α-YY1 rabbit polyclonal IgG (Santa Cruz, sc-281X),α-CREB-1 mouse monoclonal IgG₂ (Santa Cruz, sc-271), α-ATF-1 mousemonoclonal IgA (Santa Cruz, sc-243), α-AP2 rabbit polyclonal IgG (SantaCruz, sc-184X), or α-GATA-4 rabbit polyclonal IgG (Ip et al., 1994) wasincubated with the indicated nuclear extract at 4° C. for 20 minutesprior to the binding reaction as described previously (Ip et al., 1994).

Plasmids

To assess the function of each of the six nuclear protein binding sitesidentified within the SM22α promoter, a series of SM22α mutantpromoter-luciferase reporter plasmids were generated by PCR™-mediatedsite directed mutagenesis as described previously (Morrisey et al.,1996). The rous sarcoma virus (RSV) LTR-driven luciferase reporterplasmid, pRSVL, and the pMSVβgal reference plasmid have been describedpreviously (Parmacek et al., 1992). The promoterless pGL2-Basic plasmid(Promega, Madison, Wis.) served as the cloning backbone for all of theluciferase reporter plasmids described herein. The p-5000/I1SM22lucplasmid, contains 5-kb of SM22α 5' flanking sequence, the untranslatedSM22α first exon, the SM22α first intron and the first 12-bp of exon 2of the SM22α gene subcloned 5' of the luciferase reporter gene. It wasconstructed by first subcloning the 8.5 kb BamHI/HindIII SM22α genomicsubfragment (containing 5-kb of 5' flanking sequence, exon 1 and 3.5-kbof intron 1) into BglII/HindIII digested pGL2-Basic vector. Next, a488-bp PCR™-generated HindIII-linkered SM22α genomic subfragment,including at its 5' end the SM22α intron 1 HindIII restriction site (seeFIG. 5A), and running to bp +76 of the SM22 cDNA (which includes 12-bpof exon 2) was subcloned into the HindIII-digested vector and itscorrect orientation (5' to 3' relative to the luciferase reporter gene)confirmed by DNA sequence analysis. The p-5000SM22luc plasmid,containing 5-kb of SM22α 5' flanking sequence subcloned 5' of theluciferase reporter gene, was constructed by first subcloning the 2.2-kbBamHIlEcoRI SM22α genomic subfragment (corresponding to bp -5000 to-2800) into BamHI/EcoRI-digested pBluescript IIKS (Stratagene La Jolla,Calif.). Next, the 1250-bp EcoRl/NcoI SM22α genomic subfragmentcorresponding to bp -1338 to -89 and the 130-bp PCR™-generated genomicsubfragment containing bp -88 (including the NcoI site at its 5' end) to+41 (including a HindIII linker at its 3' end) was ligated into theEcoRI/HindIII-digested vector. Then, the 1.4-kb EcoRI SM22α genomicsubfragment (corresponding to bp -2800 to -1339) was subcloned into theEcoRI-digested plasmid and its orientation confirmed by DNA sequenceanalysis. Finally, the resulting SM22α genomic subfragment correspondingto bp -5 kb to +41 was excised from the Bluescript phagemid with BamHIand HindIII and subcloned into BglII/HindIII-digested pGL2-Basic. Thep-1338SM22luc plasmid containing the 1379-bp SM22α genomic subfragment(bp -1338 to +41) subcloned 5' of the luciferase reporter in thepGL2-Basic vector, was constructed using the 1250-bp EcoRI/NcoI SM22αgenomic subfragment (bp -1338 to -89) and the 130-bp (bp -88 to +41)PCR™-generated genomic subfragments described herein. The p-441SM22lucplasmid contains the 482-bp (bp -441 to +41) PstI/HindIII SM22α genomicsubfragment subcloned into BglII/HindIII-digested pGL2-Basic plasmid.The p-300SM22luc and p-162SM22luc luciferase reporter plasmids,respectively, contain the PCR™-generated bp -300 to +41, and -162 to +41SM22α genomic subfragments (including synthetic XhoI (5' end) andHindIII (3' end) linkers), subcloned into XhoI/HindIII-digestedpGL2-Basic vector. All PCR™-generated genomic subfragments wereconfirmed by dideoxy DNA sequence analysis.

The following SM22α mutant promoter-luciferase reporter plasmids weregenerated and named according to the specific nuclear protein bindingsite (or sites) within the promoter that was mutated (mutatednucleotides within each nuclear protein binding site are underlined):

p-441 SM22μSME-1

5' AAGGAAGGGTTTCAGGGTCCTGCCCATAGATCTTTTTTCCCGGCCGC 3' (SEQ ID NO:38);p-441SM22μSME-2

5' CCGCCCTCAGCACCGCGGATCCCCGACCCCCGCAGCATCTCCG 3' (SEQ ID NO:39);p-441SM22μSME-3 5' CTCGGATCCATGCTAGCAATGAATTCGGCTGCCCCCG b 3' (SEQ IDNO:40); p-441 SM22μSME-4

5' TCCAACTTGGTGTCTTTCCCCAAGGATCCAGCCTGTGTGGAGTG 3' (SEQ ID NO:41); p-441SM22μSRF/SME-4

5' TCCAACTTGGTGTCTTTCCCCGGATATGGAGCCTGTGTGGAGTG 3' (SEQ ID NO:42); p-441SM22μYY1/SME-4

5' TCCAACTTGGTGTCTTTCCCCAAATAGGAGCCTGTGTGGAGTG 3' (SEQ ID NO:43);

p-441SM22μSME-5 5' GGGCAGGTACCGAATTCAGCG 3' (SEQ ID NO:44); p-441SM22μCREB/SME-6

5' GGACGGCAGAGGGGATCCATGCCTGCCTAGGCGGCCG 3' (SEQ ID NO:45);

p-441 SM22μYY1/SME-6 5' GGACGGCAGAGGGGATCCATCACTGCCTAGGCGGCCG 3' (SEQ IDNO:46). In addition, several SM22α promoter-luciferase reporter plasmidswere subcloned that contain mutations in two cis-acting sequences in theSM22α promoter sequence. p-441SM22μCArG contains the mutations describedabove in the SME-1 and SME-4 sites, and p-441 SM22μSME2/5 contains themutations described above in the SME-2 and SME-5 sites. EachPCR™-generated SM22α promoter mutant was confirmed by DNA sequenceanalyses as described previously (Parmacek et al., 1992).

To identify functionally important cis-acting elements that control theexpression of the SM22α gene in vivo, four transgenic vectors werecloned each of which encodes the bacterial lacZ reporter gene under thetranscriptional control of the native or mutated SM22α promoterfragments. The p-5000SM22-lacZ, p-441SM22-lacZ plasmid, thep-441SM22μCArG-lacZ, and p-280SM22-lacZ plasmids, contain the 5-kb SM22αpromoter, the 441-bp SM22α promoter, the 441-bp SM22α promoter withmutations in SME-1 and SME-4 (that abolish binding of SRF), and the280-bp SM22α promoter, respectively, subcloned immediately 5' of thebacterial lacZ reporter gene in a modified pBluescript IIKS (Stratagene)plasmid.

Transfections and Luciferase Assays

1×10⁶ passage three primary rat aortic SMCs, C2C12 myotubes and A7r5cells, respectively, were split and plated 24 hours prior totransfection and transfected with either 50 or 100 μg of Lipofectinreagent (Life Technologies, Gaithersburg, Md.), 15 μg of luciferasereporter plasmid and 5 μg of the pMSVβgal reference plasmid as describedpreviously (Parmacek et al., 1992; Ip et al., 1994; Solway et al., 1995;Samaha et al., 1996). 1×10⁶ NIH 3T3 or COS-7 were transfected with 20 μgof Lipofectin reagent, 15 μg of the luciferase reporter plasmid and 5 μgof the pMSVβgal reference plasmid as described previously (Ip et al.,1994; Forrester et al., 1991). 1×10⁶ Hep G2 cells were transfected using360 μg of Lipofectamine reagent (Life Technologies, Gaithersburg, Md.),26 μg of luciferase reporter plasmid and 9 μg of the pMSVβgal referenceplasmid. Following transfection, cell lysates were prepared, normalizedfor protein content and luciferase and β-galactosidase assays wereperformed as described previously (Parmacek et al., 1992). All studieswere repeated at least three times to assure reproducibility and permitthe calculation of standard errors. Luciferase activities (light units)were corrected for variations in transfection efficiencies as determinedby assaying cell extracts for β-galactosidase activities. Data areexpressed as normalized light units ±S.E.M.

Transgenic Mice

Transgenic mice were produced harboring the p-5000SM22-lacZ,p-441SM22-lacZ, p-441SM22μCArG-lacZ and p-280SM22-lacZ transgenesaccording to standard techniques as described previously (Metzger etal., 1993). To identify transgenic founder mice, Southern blot analysiswas performed using the radiolabeled lacZ probe and high molecularweight DNA prepared from tail biopsies of each potential founder. Thenumber of copies per cell were quantitated by comparing thehybridization signal intensity (DPM) to standards corresponding to 1, 10and 100 copies/cell using a Molecular Dynamics PhosphorImager™. At leastfour independent founder lines containing each transgene were identifiedas described previously (Parmacek and Leiden, 1989). Transgenic embryos(less than ED 15.5) and tissue sections from adult mice were fixed,stained for β-galactosidase activity and counter-stained withhematoxylin and eosin as described previously (Lin et al., 1990). Ofnote, 0.02% NP-40 was added to PBS during the fixation of whole mountembryos. In addition, to visualize the arterial system of mouse embryos,following staining for β-galactosidase activity, embryos were dehydratedin methanol for 24 h and cleared in 2:1 (V/V) benzyl benzoate:benzylalcohol for 2 h.

EXAMPLE 1 Isolation and Structural Characterization of the Murine SM22αcDNA

Murine SM22α cDNA clones were isolated using the polymerase chainreaction in conjunction with synthetic oligonucleotide primers derivedfrom the previously published sequence of the rat SM22α cDNA (Nishida etal., 1993). The nucleotide sequence of the full-length murine SM22α cDNAis designated herein as SEQ ID NO:8. The murine SM22α cDNA encodes a201-amino acid polypeptide, SEQ ID NO:9, with a predicted molecular massof 22.5 kDa. It is composed of a 76-bp 5' untranslated region, a 603-bpopen reading frame, and a 403-bp 3' untranslated region. Of note, 23-bp5' of the poly(A) tail there is an A/T rich sequence (AATATA) which mayfunction as the polyadenylation signal.

A comparison of the coding sequences of the murine and human SM22α cDNAs(Shanahan et al., 1993) demonstrated that the two sequences are 91% and97% identical at the nucleotide and amino acid levels, respectively. Inaddition, a comparison of the coding sequences of the murine SM22α cDNAand the murine smooth muscle thin filament regulatory protein, calponin(Strasser et al., 1992), demonstrated that these two sequences are 23%identical and 32% conserved at the amino acid level. Interestingly, theprotein sequence encoded by the murine SM22α cDNA exhibits partialsequence identity with the sequence of the Drosophila muscle proteinmp20(Lees-Miller et al., 1987) across the entire cDNA, suggesting thatthese two proteins may have evolved from a common ancestral gene. Twodomains were particularly well conserved between these proteins. Onedomain with 14/19 amino acid identity (corresponding to amino acids104-122 of the murine SM22α protein) may represent a calcium bindingdomain oriented in an EF hand conformation (Kretsinger, 1980). Thesecond C-terminal conserved domain with 13/24 amino acid identity(corresponding to amino acids 158-181 of the murine SM22α protein) is adomain of unknown function.

SM22α Is Encoded by a Single Copy Gene

The finding of a putative calcium binding domain oriented in an EF handconformation suggested that SM22α might be related to other members ofthe troponin C supergene family of intracellular calcium bindingproteins including slow/cardiac troponin C, fast skeletal troponin C,calmodulin, myosin light chain and parvalbumin (Kretsinger, 1980). Inorder to determine whether SM22α is encoded by a single copy gene in themurine genome and whether SM22α is related to other troponin C supergenefamily members, the murine SM22α cDNA was used to probe Southern blotscontaining murine genomic DNA under both high and low stringencyconditions. Under high stringency conditions, the murine SM22α cDNAprobe hybridized to one or two BamHI, EcoRI, HindIII, PstI and XbaIbands, suggesting that SM22α is a single copy gene in the murine genome.Interestingly, no additional bands were demonstrated under lowstringency conditions, suggesting that although the SM22α gene may haveone EF hand calcium binding domain, it is not closely related to othermembers of troponin C supergene family.

EXAMPLE 2 Expression of the SM22α Gene

Previous studies have suggested that SM22α protein is expressed solelyin smooth muscle-containing tissues of the adult and may be one of theearliest markers of the smooth muscle cell lineage (Gimona et al., 1992;Duband et al., 1993; Nishida et al., 1993). To determine the in vivopattern of SM22α gene expression, the SM22α cDNA was hybridized toNorthern blots containing RNAs prepared from 12-week old murine tissues.The murine SM22α cDNA probe hybridized to one predominant mRNA speciesof approximately 1.2-kb. SM22α mRNA is expressed at high levels in thesmooth muscle-containing tissues of aorta, small intestine, lung, spleenand uterus. In addition, prolonged autoradiographic exposures revealedvery low, but detectable, levels of SM22α mRNA in heart, kidney,skeletal muscle and thymus.

In order to determine the cell-specificity of SM22α gene expression, theSM22α cDNA probe was hybridized to northern blots containing RNAsprepared from rat aortic vascular SMCs, the rat SMC line A7r5, murineNIH 3T3 and C3H10T1/2 fibroblasts, the SV40-transformed monkey kidneycell line COS-7, murine C2C12 myoblasts and myotubes, the humanhepatocellular carcinoma cell line Hep G2 and the murine lymphoid cellline EL4. High levels of SM22α MRNA were detected in primary rat aorticvascular SMCs and the smooth muscle cell line A7r5. Of note, detectionof a second 1.5 kb species of mRNA represents cross hybridization of theSM22α probe to the murine calponin mRNA. In addition, SM22α mRNA wasexpressed in both undifferentiated C2C12 myoblasts andterminally-differentiated C2C12 myotubes. Finally, a faint hybridizationsignal was detectable in NIH 3T3, C3H10T1/2, and Hep G2 cells after a3-day autoradiographic exposure. Quantitative PhosphorImager™ analysisof these low level hybridization signals revealed that SM22α mRNA isexpressed in these three non-myogenic cell lines at less than 1.5% theintensity of SM22α gene expression in A7r5 and primary SMCs. Thus, inaddition to primary SMCs and SMC lines, SM22α mRNA is expressed in otherembryonic skeletal muscle cell lineages such as C2C12 myoblasts andmyotubes, but not in other non-myogenic cell lineages.

SM22α Is Expressed in Both Cell Cycle Arrested and Proliferating SMCs

Within the tunica media of the arterial wall the vast majority ofvascular SMCs are maintained in a non-proliferating, quiescent state andexpress contractile proteins (Owens et al., 1986; Rovner et al., 1986;Taubman et al., 1987; Ueki et al., 1987; Gimona et al., 1990; Shanahanet al., 1993; Ross, 1993; Forrester et al., 1991). However, in responseto vascular injury, SMCs migrate from the tunica media to the intimallayer, proliferate and assume a "synthetic phenotype" (Ross, 1986;Schwartz et al., 1986; Zanellato et al., 1990; Ross, 1993; Forrester etal., 1991; Schwartz et al., 1992; Liu et al., 1989). Previous studieshave demonstrated that many genes encoding vascular SMC contractileproteins are down-regulated during this process (Owens et al., 1986;Rovner et al., 1986; Ueki et al., 1987; Gabbiani et al., 1981). Thus,the SM22α gene may be unique in that its expression is notdifferentially regulated during progression through the cell cycle. Inorder to address this question, cultures of low passage number primaryrat aortic SMCs were synchronized in the G₀ /G₁ stage of the cell cycleby serum starvation for 72 hrs. FACS analyses revealed that under theseconditions approximately 90% of cells are arrested in G₀ /G₁ (Chang etal., 1995). The cells were then serum-stimulated and RNA was preparedfrom replicate cultures at the time of serum stimulation (t₀), and at 8hrs, 12 hrs, 16 hrs, and 24 hrs post-stimulation. After serumstimulation, the arrested vascular SMCs begin to pass through the G₁ /Scheckpoint of the cell cycle at approximately 12 hrs and by 24 hrspost-stimulation greater than 50% of cells are in the S and G₂ /M phasesof the cell cycle (Chang et al., 1995). A northern blot analysisdemonstrated no differences in SM22α gene expression in cell cyclearrested versus proliferating SMCs as assessed by quantitativePhosphorolmager™ analysis of the hybridization signal. Thus, in contrastto other smooth muscle contractile proteins, such as smooth musclemyosin heavy chain (Rovner et al., 1986), smooth muscle α-actin (Owenset al., 1986) and calponin, SM22α appears to be constitutively expressedat high levels in both quiescent and proliferating vascular SMCs.

EXAMPLE 3 Isolation and Structural Characterization of a SM22α GenomicClone

A full length murine SM22α genomic clone of 20-kb was isolated byscreening a murine 129SV genomic library with a SM22α cDNA probe underhigh stringency conditions. Exons were identified by hybridization withspecific cDNA fragments and their boundaries confirmed by DNA sequenceanalysis. The nucleic acid sequence of the genomic clone is designatedherein as SEQ ID NO:1, containing exon 1, SEQ ID NO:2, containing exons2, 3 and 4, and SEQ ID NO:6, containing exon 5. There is approximately a4 kb gap between SEQ ID NO:1 and SEQ ID NO:2, and approximately a 450base gap between SEQ ID NO:2 and SEQ ID NO:6. The amino acid sequencesare encoded by exons 2, 3 and 4 and are designated herein as SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:7. The murine SM22α gene iscomposed of five exons spanning 6.2-kb of genomic DNA.

The transcriptional start site of the SM22α gene was identified by RNaseprotection, primer extension and 5' RACE PCR™ analyses. Primer extensionanalyses utilizing an antisense synthetic oligonucleotide correspondingto bp 80-104 of the SM22α cDNA resulted in a major extended product of104-bp (arrow) which was generated at reaction temperatures up to 56° C.In addition, 5' RACE PCR™ was performed utilizing an antisenseoligonucleotide primer corresponding to bp 234-258 of the SM22α cDNA.DNA sequence analyses of eight random 5' RACE clones revealed atranscriptional start site 76-bp 5' of the initiation codon in seven ofeight clones and 72-bp 5' of the initiation codon in one of eightclones. RNase protection analyses were also performed using an antisensecDNA probe corresponding to bp -88-+44 of the SM22α genomic sequence asdeduced by DNA sequence and Southern blot analyses. These analysesrevealed a major protected fragment of 44-bp (arrow) corresponding to atranscriptional start site 76-bp 5' of the initiation codon. Inaddition, a second, minor (20% relative signal intensity) protectedfragment of 54-bp was also demonstrated. Taken together, these dataallowed the identification of the major transcriptional start site ofthe murine SM22α gene 76-bp 5' of the initiation codon.

The complete coding sequence and 1339-bp of 5' flanking sequence of theSM22α gene was determined and each of the splice junctions conforms tothe consensus splice donor-acceptor patterns as described by Breathnachand Chambon (Breathnach et al., 1981). In order to identify potentialtranscriptional regulatory elements, 1339-bp of 5' sequence flanking thecap site was searched for a variety of transcriptional regulatoryelements using MacVector DNA sequencing software (Kodak/IBI). Thenucleotide sequence TTTAAA, which might function as a TATA box waspresent 29-bp 5' of the start site. A consensus CAAT box was notidentified in the immediate 5' flanking region of the SM22α gene. Acomputer homology search for previously described muscle-specific and/orskeletal or cardiac muscle lineage-restricted transcriptional regulatoryelements revealed five consensus E boxes/bHLH myogenic transcriptionfactor binding sites (CANNTG [Olson, 1990; Tapscott et al., 1991; Lassaret al., 1989]) located at bps -534, -577, -865, -898, -910, and -1267,three consensus GATA-4 binding sites (WGATAR [Evans et al., 1988])located at bps -504, -828, -976, and two AT-rich, potential MEF-2/rSRFbinding sites (YTAWAAATAR, SEQ ID NO:13 [Gossett et al., 1989]) locatedat bps -407 (TTtAAAATcG, SEQ ID NO:14, small letters denote mismatchesfrom the consensus MEF-2 sequence) and -770 (TTcAAAATAG, SEQ ID NO:15).In addition, functionally important nuclear protein binding sites whichhave been identified in previously characterized skeletal andcardiac-specific transcriptional regulatory elements included twoconsensus CArG/SRF binding sites (Minty et al., 1986) located at bps-150 and -273 and one CACC box (Dierks et al., 1983) located at bp -104.Finally, four AP2 (CCCMNSSS, SEQ ID NO:16 [Mitchell et al., 1987]), oneSp1 (KRGGCKRRK, SEQ ID NO:17 [Dynan et al., 1983]), and two NF-IL6(TKNNGNAAK, SEQ ID NO:18 [Akira et al., 1990]) binding sites werelocated in the 5' flanking region.

EXAMPLE 4 Identification of the cis-Acting Transcriptional RegulatoryElements That Control SM22α Gene Expression

In order to identify the functionally important cis-acting sequencesthat regulate transcription of the SM22α gene in SMCs, a series oftransient transfections were performed using SM22α-luciferase reporterconstructs and primary rat aortic vascular SMCs and the SMC line, A7r5,both of which express high levels of SM22α mRNA. Transfection of A7r5cells with the plasmid p-5000/I1SM22luc, containing 5-kb of 5' flankingsequence and the entire 4-kb SM22α intron 1 sequence (the initiationcodon is located in exon 2), resulted in a 250-300-fold induction inluciferase activity as compared to the promoterless control plasmid,pGL2-Basic (FIG. 1A, lanes 1 and 2). This level of transcriptionalactivity was comparable to that obtained following transfection of A7r5cells with the RSV-containing luciferase reporter plasmid, pRSVL (FIG.1A, lanes 2 and 8). In order to determine whether this transcriptionalactivity was due to the immediate 5' flanking region of the SM22α gene,or alternatively, was due to a transcriptional regulatory elementlocated within the first intron of the SM22α gene, the activities of thep-5000/I1SM22luc and p-5000SM22luc plasmid were compared (FIG. 1A, lanes2 and 3). Transfection of A7r5 cells with the p-5000SM22luc plasmid,containing only 5-kb of 5' flanking sequence, resulted in high-leveltranscription of the luciferase reporter gene comparable (on a molarbasis) to levels obtained with the p-5000/I1SM22luc plasmid. Thus, the5' flanking region of the SM22α gene contains cis-acting sequenceelements required for high-level transcription in A7r5 cells.

To further localize the 5' flanking elements of the SM22α gene thatdirect high-level expression in SMCs, a series of 5' deletion mutantswere transfected into both A7r5 cells (FIG. 1A) and primary cultured rataortic vascular smooth muscle cells (FIG. 1B). In both A7r5 cells andprimary vascular SMCs, the p-441SM22luc plasmid, containing 441-bp of 5'flanking sequence, increased transcription of the luciferase reporter tolevels comparable to the p-5000SM22luc plasmid and the p-1338SM22lucplasmids (FIG. 1A lanes 3, 4, 5 and FIG. 1B lanes 2, 3). However,transfection of both A7r5 cells and primary vascular SMCs with theluciferase reporter plasmids p-300SM22luc and p-162SM22luc containing300-bp and 162-bp, respectively, of 5' flanking sequence resulted in 50%and 90% reductions in normalized luciferase activities as compared withthose obtained with the p-441SM22luc plasmid (FIG. 1A lanes 5, 6, 7 andFIG. 1B lanes 3, 4, 5). These data demonstrated that 441-bp of SM22α 5'flanking sequence, containing the endogenous SM22α promoter, issufficient to direct high-level transcriptional activity in both A7r5cells and primary rat aortic SMCs.

EXAMPLE 5 Cellular-Specificity of the SM22α Promoter

In order to characterize the cellular-specificity of the SM22α promotersequence, the transcriptional activities of the 441-bp SM22α promotercontaining plasmid, p-441 SM22luc, was compared to the positive controlplasmid containing the rous sarcoma virus LTR, pRSVL, in primary ratvascular SMCs, the smooth muscle cell line A7r5, NIH 3T3 fibroblasts,COS-7, and Hep G2 cells. Consistent with the lineage-restricted patternof SM22α mRNA expression demonstrated in these cell lines, thepromoter-containing plasmid, p-441SM22luc, was active in primary rataortic SMCs and A7r5 cells, increasing transcription of the luciferasereporter gene approximately 2500-fold and 540-fold, respectively, overthat induced by transfection with the promoterless pGL2-Basic plasmid(FIG. 2). This level of promoter activity was comparable to levelsobtained following transfection of these cells with the RSV LTR-drivenpositive control plasmid (FIG. 2). In contrast, the 441-bp SM22αpromoter was inactive in NIH 3T3, COS-7 and Hep G2 cells (FIG. 2).

DNA sequence analyses revealed that this 441-bp promoter contains twoCArG/SRF boxes (Minty et al., 1986), a CACC box (Dierks et al., 1983),and one A/T-rich, potential MEF-2/rSRF binding site (Gossett et al.,1989), cis-acting elements which have each been demonstrated to beinvolved in the transcriptional programs that regulate skeletal andcardiac muscle-specific gene expression. However, unlike most previouslydescribed skeletal muscle-specific transcriptional regulatory elements,this sequence lacked a canonical E box binding site for the myogenicbHLH transcription factors (Tapscott et al., 1991; Lassar et al., 1989).Thus, the endogenous 441-bp SM22α promoter contains all of thecis-acting sequence elements required to recapitulate the smooth musclelineage-restricted pattern of SM22α gene expression demonstrated invivo.

EXAMPLE 6 Generation of SM22α-βgal Transgenic Mice

A reporter construct was first prepared in which the 441-bp minimalSM22α promoter was subcloned immediately 5' of the bacterialβ-galactosidase reporter gene (lacZ). The transgenic vector wasgenerated from a pBluescript-KS phagemid containing AscI restrictionsites flanking the polylinker sequence. This construct is referred toherein as -441SM22lacZ. The transgene was microinjected into oocytesthat were transplanted into pseudo-pregnant hosts as described inMetzger et al., 1993 (incorporated herein by reference). To identifytransgenic founder mice, Southern blot analysis was performed using theradiolabeled lacZ probe and high molecular weight DNA prepared from tailsnips of each potential founder pup. The radiolabeled lacZ cDNA probehybridized to the expected 4.2 kb BamHI-digested band in 4 of 17 pupsanalyzed. The four founders contained between 5 and 160 copies per cellas assessed by comparing the hybridization signal intensity (DPM) tostandards corresponding to 1, 10 and 100 copies per cell using aMolecular Dynamics PhosphorImager™.

The F1 -441 SM22lacZ #14 male was crossed with a CD-1 female and E11.5embryos from this litter were isolated, genotyped (using PCR™), fixedand stained for β-galactosidase activity. Transgenic embryos were easilydistinguished from their non-transgenic litter mates by the obvious bluestaining along their distal somites. This pattern correlated with thetransient pattern of SM22α gene expression observed in the developingsomites. In ED11.5 embryos, the endogenous SM22α gene is expressedthroughout the primitive heart tube, developing somites, dorsal aortaand the forming branch arteries (Li et al., 1996a). Whole mount stainingof ED11.5 embryos demonstrated high level β-galactosidase activitythroughout the developing arterial system. Blue staining was observedthroughout the dorsal aorta, the carotid and vertebral arteries, thecerebral arteries, the umbilical arteries and the aortic arches. A highpower section through the iliac artery, demonstrated that expression ofthe lacZ transgene was restricted to 1-2 layers of cells underlying thearterial endothelium. In addition, β-galactosidase activity was detectedwithin the myotomal component of the developing somites and within thebulbo-truncus region (future outflow tract) and at low levels within thebulbo-cordis region (future right ventricle) of the primitive heart.β-galactosidase activity was not detected within the future leftventricle, left atrium or right atrium at this stage of embryonicdevelopment. Surprisingly, although the SM22α gene is expressed at highlevels in smooth muscle cells lining the pulmonary airways, as well aswithin the gastrointestinal and genitourinary tracts, no β-galactosidaseactivity was detected in the developing lung buds, gastrointestinalmucosa, or the uterine or bladder mucosa during late murineembryogenesis or postnatal development. Thus, the 441 bp SM22α promoteris necessary and sufficient to activate transcription in vascular SMC'sin a lineage-restricted fashion in transgenic mice. In addition, thisminimal promoter element contains cis-acting sequences required toactivate transcription of the SM22α gene in the developing somites.These data also demonstrate that SM22α gene expression is regulated atthe level of transcription.

It is noteworthy that the normalized luciferase activity obtained withthe 300-bp promoter was still 100-fold above that obtained withpromoterless control plasmids in these transient transfection assays. Todetermine whether a 280-bp SM22α promoter fragment (bp -280-+41) wassufficient to direct arterial SMC-specific gene expression, theinventors produced eight independent lines of transgenic mice in whichthe lacZ gene was placed under the transcriptional control of the 280-bpSM22α promoter. These mice contained between 2 and 34 copies of thetransgene per cell. The 280-bp of 5' flanking sequence was sufficient todirect high level β-galactosidase activity (blue staining) to arterialSMCs and the myotomal component of the somites of ED11.5 mice. Of note,virtually identical patterns of transgene expression were demonstratedin 4 independent lines analyzed at ED11.5 in which copy numbers variedbetween 2 and 34 copies per cell. Interestingly, dense blue staining wasdetected within the cardiac outflow tract (a neural crest derivative)while a somewhat patchy pattern of blue staining was present in thedeveloping arterial system (which is derived from lateral mesoderm andneural crest). Higher power sections confirmed that virtually every cellwithin the cardiac outflow tract stained blue. Interestingly, dense bluestaining was detected within the mesenchymal cells that compose theaorticopulmonary spiral septum which is present at ED11.5. In addition,most, but not all, cells underlying the epithelium of the developingarteries stained blue. Taken together, these data demonstrate that the280-bp SM22α promoter is sufficient to program lineage-restrictedtranscription in arterial SMCs and the developing somites. However, incontrast to the endogenous pattern of SM22α gene expression, the 441-bp(and 280-bp) SM22α promoter does not contain the cis-acting elementsthat control SM22α transcription in either visceral (gastrointestinal,uterine, bladder, and bronchial) or venous SMCs nor in the primitiveheart tube. Finally, it is noteworthy that the inventors observedvirtually the same arterial SMC-specific pattern of expression using the5000-bp SM22α promoter in transgenic mice. These data strongly suggestthat distinct transcriptional programs distinguish tissue-restrictedsubsets of SMCs (even within the vasculature).

Xgal Tissue Staining

The lung, heart, liver, kidney, spleen, testis or ovary, and skeletalmuscle are excised from euthanized animals, and stained to revealβ-galactosidase activity. If β-galactosidase activity is evident innon-transgenic mice, the transgenic lines are generated using a nuclearlocalizing β-galactosidase isoform to minimize false-positive staining(Hughes and Blau, 1990). To reveal β-galactosidase activity, tissues arewashed in PBS, then fixed in 1.25% glutaraldehyde (lung is fixed asbelow). After washing in Ca⁺² - and Mg⁺² -free buffer, tissues areincubated overnight in the dark in Xgal solution (50 mM Tris HCl pH 7.5,2.5 mM potassium ferriferrocyanide, 15 mM NaCl, 1 mM MgCl₂, 0.5 mg/mlXgal), then paraffin embedded; 4 micron sections are counterstained witheosin.

Data Analysis

The tissue and cellular distribution of Xgal staining, reflecting SM22αpromoter transcriptional activity, is recorded for each transgenic linestudied, and compared qualitatively among experimental conditions.Quantitative assessment of lung and tracheal SM22α promotertranscriptional activity is also performed by RNase protection assay forlacZ mRNA, which is compared among study groups using ANOVA followed bymultiple range testing. To test whether potential differences in lacZmRNA levels might stem from different amounts of smooth muscle amonggroups, airway smooth muscle area vs. circumference curves is comparedbetween groups as described by James et al. (James et al., 1989);pulmonary arterial area vs. circumference curves are likewise compared.

EXAMPLE 7 Expression of SM22α in Lung

SM22α mRNA by is detected in the lungs by in situ hybridization. Adigoxigenin-labeled cRNA corresponding to the reverse complement ofmouse SM22α cDNA bp 644 to 1007 was prepared by in vitro transcription(MaxiScript™ Kit, Ambion, and Genius™ 4 Kit, Boehringer Mannheim). Insitu hybridization was performed on a lung specimen obtained at autopsyfrom a patient without lung disease. Hybridized probe is detectedimmunohistochemically with an anti-dioxigenin antibody linked toalkaline phosphatase. The SM22α cRNA binds selectively to airway smoothmuscle and to pulmonary vascular smooth muscle; black anthracoticpigment was also evident in this specimen (typical of urban dwellers).

EXAMPLE 8 Adenovirus Mediated Expression of a Constitutive Rb GeneProduct

The Rb protein inhibits cell cycle progression in many mammalian celltypes (Hollingsworth et al., 1993), and has been shown to be animportant regulator of vascular smooth muscle proliferation (Chang etal., 1995). In its unphosphorylated state, the Rb gene product binds andinactivates certain cellular transcription factors that are required forcell cycle progression (Chen et al., 1989) and upon phosphorylation, thetranscription factors are released and the cell progresses through theproliferation cycle. A gene encoding a phosphorylation deficient Rb geneproduct has been constructed and shown to constitutively inhibit smoothmuscle cell cycle proliferation (Chang et al., 1995) when transfectedinto rat aortic smooth muscle cells in a replication defectiveadenovirus vector. Further, the Chang et al. (1995) reference also showsthat replication deficient adenovirus vectors can be used to expressheterologous genes in rat carotid arteries in vivo upon direct exposureof isolated segments of injured artery to the adenovirus. A similarstudy was done in isolated porcine arteries and again the adenoviraltransferred constitutive Rb gene product was shown to be expressed andto inhibit smooth muscle cell proliferation.

The inventors propose that this Rb gene product may also be expressedunder the control of the smooth muscle specific promoter disclosedherein, thus directing expression of the Rb gene product specifically insmooth muscle cells. This offers the advantage of administration of thevirus vector by a less invasive method such as intravenous injection. Itis also contemplated that other cell cycle control gene products, suchas p53 for example, would be effective in this method of preventingrestenosis.

EXAMPLE 9 Identification of Smooth Muscle Specific Trans-ActingTranscription Factors

Identification of Nuclear Protein Binding Sites in the SM22α Promoter

To identify nuclear protein binding sites within the 441-bp SM22αpromoter, DNase I footprint analyses were performed with nuclearextracts prepared from the SMC line, A7r5 (which express high levels ofSM22α mRNA (Solway et al., 1995)), and the non-muscle cell line, NIH3T3. Six nuclear protein binding sites were identified on both the senseand antisense strands with nuclear extracts prepared from the SMC line,A7r5. The six nuclear protein binding sites were designated smoothmuscle elements (SME)-1-6, respectively (FIG. 3D). Two footprintedregions, SME-1 (bp -279 to -256) and SME-4 (bp -171 to -136), containembedded SREs, or CArG boxes (CCWWWWWWGG, SEQ ID NO:47) (FIG. 3D, grayboxes embedded in SME-1 and SME-4), that have been shown previously tobind the MADS box transcription factor, SRF, and play an important rolein regulating transcription of the genes encoding skeletal and cardiacα-actin (Minty and Kedes, 1986; Moss et al., 1994; Muscat et al., 1992).Of note, fine differences in the digestion patterns between nuclearextracts prepared from A7r5 and NIH 3T3 cells could be distinguishedover the SME-4 site (FIG. 3B, compare lanes 4-5 and 6-7; FIG. 3B,compare lanes 11-12 and 13-14). Several studies suggest that nucleotidesembedded within and/or flanking CArG boxes regulate binding of ternarycomplex factors (TCFs), including members of the ets and homeodomainfamilies of transcription factors. Thus, the finding that a PEA3 motif(bp -295 to -289), which has been demonstrated to bind in vitro to etsfamily members, lies 23-bp 5' of the SME-1 motif is noteworthy.Similarly, SME-4 spans a GGAG motif (bp -142 to bp -139) which has beendemonstrated to bind to TCFs in the ets family of transcription factors(Johansen and Prywes, 1995). Moreover, the SME-4 motif contains theembedded motif ATATGG (bp -146 to bp -141) which has been demonstratedto bind homeobox transcription factors including Csx/Nkx2.5 (Chen etal., 1996).

The SME-2 nuclear protein binding site (bp -249 and bp -216) containsconsensus binding motifs for the ubiquitously expressed transcriptionfactors, Sp1 (KRGGCKRRK) and AP2 (CCCMNSSS) (FIG. 3D, SME-2, grayboxes). Fine differences in the digestion patterns between nuclearextracts prepared from A7r5 and NIH 3T3 cells could be distinguishedover this site (FIG. 3B, compare lanes 4-5 and 6-7; FIG. 3B, comparelanes 11-12 and 13-14). The SME-3 nuclear protein binding site (bp -215to bp -186), which is flanked by DNase I hypersensitive sites at bothits 5' and 3' borders, was protected only by nuclear extracts preparedfrom A7r5 and not by extracts prepared from NIH 3T3 cells (FIG. 3B,compare lanes 4-5 and 6-7; FIG. 3B, compare lanes 11-12 and 13-14). Thisnuclear protein binding site has not been described previously. TheSME-5 nuclear protein binding site (bp -86 to bp -66) once againcontains consensus Sp1 and AP2 motifs (FIG. 3C). The SME-6 nuclearprotein binding site (bp -59 to -35), lies immediately 5' of thenon-consensus TATA box (TTTAA), and contains nucleotide sequences thathave been demonstrated previously to bind the cyclic AMP responseelement (CRE) binding proteins (for review see (Lalli and Sassone-Corsi,1994)) (FIG. 3C). Of note, an AT-rich sequence (bp -408 to -415) with8/10 bp sequence identity with the consensus MEF2 binding motif (Gossettet al., 1989) was not protected with either A7r5 or NIH 3T3 nuclearextracts. Taken together, these studies demonstrated six nuclear proteinbinding sites within the murine SM22α promoter (FIG. 3D). Three of thesebinding sites (SME-2, SME-3 and SME-4) demonstrated differentialpatterns of digestion when incubated with nuclear extracts prepared formA7r5 and NIH 3T3 cells.

Characterization of Trans-Acting Factors That Bind to the SM22α Promoter

To assess the number, specificity, and identity, of nuclear proteinsthat bind to the arterial SMC-specific SM22α promoter, a series ofelectrophoretic mobility shift assays (EMSAs) were performed. Todetermine whether the SME-1/CArG and SME-4/CArG bind common,overlapping, or distinct, sets of trans-acting factors, EMSAs wereperformed using radiolabeled SME-1 and SME-4 oligonucleotide probes. Theradiolabeled SME-1 oligonucleotide probe bound three specific nuclearprotein complexes, designated A-C (FIG. 4A, lane 2), as determined byaddition of specific and non-specific unlabeled competitoroligonucleotides to the binding reactions (lanes 2-4). Of note,unlabeled SME-4 oligonucleotide competed for binding of complex A, butfailed to compete for complexes B and C (FIG. 4A, lanes 5 and 6).Unlabeled Sp1 oligonucleotide competed for binding of complex B (thatco-migrated with complex A), as well as, complex C (FIG. 4A, lanes 7 and8). Antibody supershift studies confirmed that complex A (arrow)contains SRF (or an antigenically related protein) and complex B (arrow)contains Sp1 (or an antigenically related protein) (FIG. 4A, lanes9-12).

EMSAs performed with the radiolabeled SME-4 oligonucleotide probedemonstrated four specific nuclear protein complexes, designated A-D(FIG. 4B, lane 2), as determined by addition of specific andnon-specific competitor oligonucleotides (lanes 3-4 and 7-8). Additionof unlabeled SME-1 oligonucleotide competed only for binding ofcomplexes A and B (FIG. 4B, lanes 5 and 6). Antibody supershift studiesrevealed that both of these low-mobility nuclear protein complexescontained a protein identical, or antigenically-related, to SRF (FIG.4B, lane 10, arrow, SRF), while complexes C and D contained a proteinidentical, or antigenically-related, to YY1 (FIG. 7B, lane 11, arrow,YY1). Taken together, these data demonstrate that, as expected, SRF (oran SRF-containing protein complex) binds to both the SME-1 and SME-4sites. Of note, the demonstration of two low mobility SME-4 bindingactivities containing SRF (FIG. 4A, complexes A and B) suggests thatone, or both, of these complexes may contain additional trans-actingfactors. In addition, SME-1 bound Sp1 (FIG. 4B, complex B) and onepotentially novel nuclear protein complex (FIG. 4B, complex C) that doesnot bind to SME-4. Conversely, SME-4 binds the ubiquitously expressedand potentially negative regulatory factor, YY1 (Gualberto et al., 1992;Lee et al., 1992; Lee et al., 1994) (FIG. 4A, complexes C and D), whileSME-1 does not.

Both the SME-2 and SME-5 sites are GC-rich motifs that contain potentialSp1 and AP2 motifs (FIG. 3D). EMSAs performed with nuclear extractsprepared from primary rat aortic SMCs and radiolabeled oligonucleotidescorresponding to the SME-2 and SME-5 (FIG. 5A) nuclear protein bindingsites, respectively, revealed identical band-shift patterns suggestingthat these two motifs might bind a common set of trans-acting factors.Each probe bound three specific nuclear protein complexes, designatedA-C (FIG. 5A, lane 2), as determined by addition of unlabeled specificand non-specific oligonucleotide competitors (FIG. 5A, lanes 6 and 9).Unlabeled SME-2 oligonucleotide competed for binding of each nuclearprotein complex that bound the radiolabeled SME-5 probe and visa versa(FIG. 5A, lane 8). Moreover, an oligonucleotide containing a consensusSp1 motif competed for binding of complexes A-C (FIG. 5A, lane 7).Antibody supershift studies revealed that complex A (arrow) was ablatedand supershifted (dashed arrow) by pre-incubation with Sp1-specificantiserum (FIG. 5A, lane 3), but not by control murine IgG, or α-AP2antiserum (lanes 3 and 5). Each of these nuclear protein complexes werealso present in nuclear extracts prepared from non-SMC lineagesincluding the lymphoid lines, WEHI and 70Z/3 (FIG. 5A, lanes 11 and 12).These data demonstrate that the SME-2 and SME-5 nuclear protein bindingsites each bind three ubiquitously expressed nuclear protein complexes,at least one of which contains a protein that is identical, orantigenically related, to Sp1.

As discussed above, SME-3 was protected from DNase I digestion bynuclear extracts prepared from A7r5 cells, but not by extracts preparedfrom NIH 3T3 cells, suggesting that this previously undescribed motifmight bind one or more SMC lineage-specific trans-acting factors. EMSAsperformed with the radiolabeled SME-3 oligonucleotide probe revealedthree specific binding activities, designated A-C (FIG. 5B, lane 2), asdetermined by addition specific and non-specific competitoroligonucleotides (FIG. 5B, lanes 3-6). Antibody supershift studiesrevealed that complex B and C contained YY1 (or an antigenically relatedprotein) (FIG. 5B, lane 10). None of the nuclear protein complexes weresupershifted by control IgG or α-Sp1 antiserum. To determine whether anyof these nuclear protein complexes were expressed in alineage-restricted fashion, EMSAs were performed with the SME-3 probeand nuclear extracts prepared from primary rat aortic SMCs, the SMCline, A7r5, C3H10Tl/2 and NIH 3T3 fibroblasts, and the mouse T cellline, EL4. Interestingly, complex C, which was ablated by pre-incubationwith a-YY1 antiserum, was present only in primary rat aortic SMCs andthe SMC line A7r5 (FIG. 5B, lane 2 and 7), but was absent in C3H10T1/2,NIH 3T3, and EL4 nuclear extracts (FIG. 5B, lanes 11-13). Moreover,three faint complexes (dashed arrows) were identified in C3H10T1/2, NIH3T3 and EL4 cells, but were not present in SMC extracts (FIG. 5C, lanes4-7). Taken together, these data suggest that the SME-3 nuclear proteinbinding site, a motif which has not been described previously, binds YY1and one or more, as yet, unidentified SMC-specific and/or lineagerestricted trans-acting factors. In addition, the radiolabeled SME-3probe binds three nuclear protein complexes that are present in severalnon-SMC lines, but not in primary vascular SMCs or the SMC line, A7r5.

EMSAs performed with a radiolabeled oligonucleotide corresponding to theSME-6 nuclear protein binding site revealed four specific nuclearprotein complexes, designated A-D, respectively (FIG. 6, lane 2). Eachof these complexes were competed with unlabeled SME-6 oligonucleotide(FIG. 6, lane 3). Moreover, addition of an unlabeled consensus CREoligonucleotide derived from the T cell receptor a enhancer competedexclusively for binding of complexes B and C (FIG. 6, lane 4).Pre-incubation of the binding reactions with α-CREB-1 antiserum ablatedand supershifted complex B (FIG. 6, lane 11, arrow, CREB-1), whilecomplex C was ablated by addition of α-ATF-1 antiserum (FIG. 6, lane 12,arrow, ATF-1). In addition, complex A (FIG. 6, arrow, Sp1) was ablatedand supershifted by pre-incubation with α-Sp1 antiserum. Finally,complex D was ablated by the addition of α-YY1 antiserum (FIG. 6, lane15, arrow, YY1). In contrast, none of the four complexes were ablated orsupershifted following pre-incubation with control rabbit or murine IgG(FIG. 6, lanes 9 and 10), or antisera that recognize GATA-4 (FIG. 6,lane 13) or SRF (FIG. 6, lane 14). Interestingly, EMSAs performed withthe radiolabeled SME-6 oligonucleotide probe and nuclear extractsprepared from the non-SMC lines, C2C12 myotubes, C3H10T1/2 and NIH 3T3fibroblasts, and EL4 T cells, revealed fine differences in themobilities of several nuclear protein complexes (and/or novelcomplexes), as well as, increased intensity in each of the SME-6 bindingactivities (FIG. 6, lanes 5-8). Taken together, these data revealed thatthe SME-6 motif binds CREB-1 and ATF-1, each of which are expressed inprimary vascular SMCs, as well as, the ubiquitously expressedtranscription factors, Sp1 and YY1.

In summary, as shown in FIG. 7, the arterial SMC-specific SM22α promotercontains six nuclear protein binding sites, designated smooth muscleelement (SME)1-6, respectively. SME-1/CArG binds SRF (and ternarycomplex factors), Sp1 and one unidentified nuclear protein complex thatis not cross-competed by SME-4/CArG oligonucleotides. SME-2 binds threespecific nuclear protein complexes at least one of which contains Sp1,each of which also binds to the SME-5 site. SME-3, a motif that has notbeen described previously, binds YY1 and two unidentified nuclearprotein complexes, one of which includes a potentially novellineage-restricted trans-acting factor. In addition, the SME-3 motifbinds several trans-acting factors which are present in nuclear extractsprepared from non-SMCs but which are not present in SMC extracts.SME-4/CArG binds nuclear protein complexes containing SRF andYY1-related proteins. Of note, two high mobility complexes were ablatedand supershifted by pre-incubation with a-SRF antiserum suggesting thatone, or both, of these nuclear protein complexes may contain accessoryfactors. Finally, SME-6 binds CREB-1, ATF-1, YY1, and Sp1.

EXAMPLE 10 Functional Characterization of the SM22α Promoter

To characterize the functional significance of each of the cis-actingelements within the SM22α promoter, specific mutations that abolishbinding of one or more trans-acting factors to nuclear protein bindingsites located within the SM22α promoter were created within the contextof the p-441SM22luc reporter plasmid. The effect of each mutation wasassessed by transient transfection analysis of each mutant SM22αpromoter luciferase reporter plasmid into primary rat aortic SMCs. Toassess the finction of the SME-1/CArG and SME-4/CArG sites, each ofwhich bind SRF, mutations were created that abolish SRF binding toSME-1, and SRF and YY1 binding to SME-4, respectively. Of note, thesemutations did not affect binding of any other nuclear protein complex(demonstrated by EMSA) to SME-1 or SME-4. Transfection analyses revealedthat mutation of the SME-1 site resulted in a 55% reduction innormalized luciferase activity compared to that obtained with thep-441SM22luc plasmid (FIG. 8A, lane 2). Remarkably, a two nucleotidesubstitution in the SME-4 site that abolished SRF binding activityresulted in a 88% reduction in normalized luciferase activity comparedto that obtained with the wild type SM22α promoter (FIG. 8A, lane 3).Moreover, the p-441SM22μCArG plasmid, which contains mutations in bothSME-1 and -4 that inhibit binding of SRF, completely abolishedtranscriptional activity of the SM22α promoter in primary rat aorticSMCs and the SMC-line A7r5 (FIG. 8A, lane 4). These data demonstratethat the SME-1 and -4 nuclear protein binding sites are required foractivity of the SM22α promoter in arterial SMCs in vitro. Moreover,these data suggest that SM22α promoter activity is critically-dependentupon the SME-4 site, SRF, and/or trans-acting factors that interact withSRF.

To assess the functional significance of each of the other (non-CArGcontaining) nuclear protein binding sites in the SM22α promoter,mutations that abolish binding of one or more trans-acting factor toeach site were created within the context of the 441-bp SM22α promotercontaining plasmid, p-441SM22luc. Because the SME-2 and SME-5 nuclearprotein binding sites, each bind a nuclear protein complex containingSp1, in addition to two other common nuclear protein complexes (FIG. 5A,lanes 5 and 7), mutations were created within the context of thep-441SM22-luc plasmid that abolish binding of each trans-acting factorto SME-2, SME-5, and both SME-2 and SME-5. Transfection of each of theseplasmids and the p-441SM22-luc plasmid into primary rat aortic SMCsdemonstrated that mutation of the SME-2, SME-5, and SME-2 and SME-5,resulted in 58%, 6% and 70% respective reductions in normalizedluciferase activities (FIG. 8B lanes 2-5). These data suggest thatwithin the context of the SM22α promoter, the SME-2, and -5 nuclearprotein binding sites are required for full promoter activity, but maybe functionally redundant.

Mutation in the SME-3 site which abolishes binding of all three SME-3binding activities (including the potentially novel lineage-restrictedtrans-acting factor) resulted in a 50% reduction in transcriptionalactivity compared to that observed with the native SM22α promoter (FIG.8B, lane 5). These data suggest either that activity of the SM22αpromoter in arterial SMCs is not critically dependent on thispotentially novel lineage-restricted trans-acting factor, oralternatively, that an additional nuclear protein binding site for thislineage-restricted trans-acting factor exists in the 441-bp SM22αpromoter (that was not detected by DNase I footprint analyses andEMSAs). To assess the functional significance of the SME-6 nuclearprotein binding site, and to determine whether the CRE located withinSME-6 is required for promoter activity, the -441 SM22μCREB/SME-6plasmid, which abolishes binding specifically of each of the CRE-relatedcomplexes (but not YY1) was compared to the p-441SM22luc reporterplasmid. The single mutation within the CREB motif reducedtranscriptional activity by approximately 60% (FIG. 8B, lane 6). Incontrast, mutations within SME-6 that do not abolish CRE bindingactivities did not significantly decrease transcriptional activities.These data suggest that CREB family members may play an importantfunctional role in transcription of the SM22α gene in VSMCs.

The Arterial SMC-Specific SM22α Promoter is CArG-Dependent in vivo

As shown above, mutations of the SME-1/CArG and SME-4/CArG elements thatinhibited binding of SRF to the SM22α promoter, totally abolished SM22αpromoter activity in arterial SMCs in vitro. To determine whether SME-1and -4 are required for activity of the SM22α promoter in arterial SMCs(and the myotomal component of the somites) in vivo, transgenic micewere produced containing a transgene, designated -441SM22μCArG, thatencodes the bacterial lacZ reporter gene under the transcriptionalcontrol of a mutant SM22α promoter containing mutations in both SME-1and SME-4 that abolish binding of SRF (as described above). Thirteenindependent -441SM22μCArG transgenic lines were produced with copynumbers ranging between 1 and 730 copies per cell. In contrast to the-441SM22lacZ transgenic mice that expressed the lacZ transgene in thearterial SMCs and within the myotomal component of the somites (FIG.1A), in 12 out of 13 independent -441SM22μCArG lines, β-galactosidaseactivity could not be detected in either the arterial SMCs or within themyotomal component of the somites at ED11.5. Of note, in one lineharboring the -441SM22μCArG transgene (that contained 5 copies percell), blue staining was detected exclusively within the cardiac outflowtract, but not within the SMCs of the dorsal aorta or branch arteries,the somites, or any other tissue. Given the low frequency at which thispattern of lacZ expression was observed, it is likely that it resultedfrom integration of the transgene near a cryptic enhancer element. Thesedata demonstrate that the SME-1 and SME-4 nuclear protein binding siteslocated within arterial SMC-specific SM22α promoter are required forSM22α promoter activity in vivo. Moreover, these data suggest stronglythat SRF plays an important role in regulating activity of the SM22αpromoter in vivo.

EXAMPLE 11 Spatial and Temporal Patterns of Activation of the ArterialSMC-Specific SM22α Promoter During Embryonic Development

The SM22α gene is expressed in the developing vasculature at least asearly as (and probably earlier than) embryonic day (ED) 9.5 in themouse, demonstrating that it is one of the earliest developmentalmarkers of the SMC lineage. As transcriptional activation precedes mRNAand protein expression, the study of SM22α provide an opportunity toexamine SMC-specific transcription at its earliest stages after (andpotentially during) migration of mesodermal stem cells from the lateralplate mesoderm and neural crest to locations throughout the embryo.Utilizing the -441SM22lacZ lines of transgenic mice, described earlier,that contain the 441-bp SM22α promoter linked to the bacterial lacZreporter gene, the spatial and temporal pattern of SM22α transcriptionalactivation during early embryogenesis, ED6, ED7.5, ED8.5, ED9.5, ED10.5,and ED11.5 is determined as confirmed by PCR™, and embryonic sectionsare fixed in 37% formaldehyde, 25% glutaraldehyde in PBS and stainedwith X-gal as previously described (Lin et al., 1990). The cells inwhich the SM22α promoter is transcriptionally active are stained blue.In these studies, the non-transgenic litter mates of the transgenic miceserve as negative controls, and these embryos are assayed forβ-galactosidase activity as described above. Sections are analyzed todetermine the spatial location and cell type in which SM22αtranscriptional activation is initially detectable (particular emphasisis placed on examining the lateral plate mesoderm and neural crest inED6, ED7.5 and ED8.5 embryos). As both cardiac myocytes and some SMCsare derived from the lateral plate mesoderm, to distinguish cardiacmyocytes from SMCs, the pattern of staining in whole mount -441SM221acZtransgenic ED6-ED9.5 embryos is compared to a staged series of embryosthat were hybridized previously to the early cardiac lineage-restrictedtranscription factor GATA-4 cRNA probe which is first detectable in thepromyocardial tissues of ED7.5 embryos (Ip et al., 1994). Takentogether, this series of studies explore the earliest stages of SMCdevelopment, and follow SMCs as they arise from lateral plate mesoderm(which gives rise to the dorsal aorta and branch arteries) and neuralcrest precursors (which give rise to the cardiac outflow tract).Moreover, because the 441-bp SM22α promoter is active exclusively withinarterial, but not visceral, SMCs these studies determine at whatembryonic stage tissue-restricted subsets of SMCs can be distinguished.

Functional Assessment of cis-acting Sequences by Transient TransfectionAnalyses

In order to determine the functional significance of each cis-actingelement within the SM22α promoter, each nuclear protein binding siteidentified by DNase I footprint analyses and characterized by EMSAs ismutated within the context of the native 441-bp SM22α promoter and thetranscriptional activity of each mutated promoter plasmid tested bytransient transfection analyses in primary rat aortic SMCs and A7r5cells. In addition, to determine whether common, overlapping, ordistinct cis-acting elements control activity of the SM22α promoter inarterial SMCs and embryonic skeletal muscle cells, C2C12 myotubes (whichexpress SM22α mRNA (Solway et al., 1995)) are also transfected asdescribed previously (Parmacek et al., 1994). Prior to mutagenesis,mutations in each nuclear protein binding site are analyzed by EMSAs toconfirm that the mutation abolishes nuclear protein complex binding.Mutations are created within the context of the p-441SM22luc plasmid bygapped heteroduplex oligonucleotide-mediated or PCR™-mediated sitedirected mutagenesis as described previously (Parmacek et al., 1994). Toassess the activity of potentially redundant elements (for example SME-1and SME-4 each bind SRF), double mutants are generated. Transienttransfections of primary rat aortic SMCs and the SMC line, A7r5, arethen performed and the luciferase activity of each mutated promoterplasmid compared to that obtained following transfection with the nativeSM22α promoter-containing luciferase reporter plasmid, p-441 SM22-luc(see FIG. 4A and FIG. 4B). The promoterless pGL2-Basic plasmid serves asa negative control and the pGL2-Control plasmid containing the SV40promoter and transcriptional enhancer serves as a positive control. Toassess transcriptional rates the results of representative luciferaseassays are confirmed by performing RNase protection assays (Solway etal., 1995) on RNA isolated from duplicate representative transfectionsusing probes complimentary to the 5' end of the firefly luciferase geneand the SM22α core promoter. In addition, transfection efficiencies aremonitored by co-transfection with the pMSVβgal reference plasmid asdescribed previously (Parmacek et al., 1992). Finally, after each of thenuclear protein binding sites located within the SM22α promoter havebeen characterized as described above, synthetic oligonucleotidescorresponding to each of the functionally important nuclear proteinbinding sites are oligomerized in various copy numbers and combinationsand inserted 5' of the minimal TATA containing SM22α promoter luciferaseplasmid, designated p-40SM22luc, in order to determine whether thepresence of randomly spaced, or oligomerized, nuclear protein bindingsites, alone or in various combinations, is sufficient for SM22αpromoter activity in arterial SMCs. These studies define a "modularpromoter" that could be utilized to target gene expression to arterialSMCs.

Analyses of SM22α Promoter Mutants in Transgenic Mice

To confirm the functional importance of cis-acting sequences within theSM22α promoter in vivo, the activity of informative SM22α promotermutants are assessed in transgenic mice. Because it is desirable todetect both qualitative (with respect to the tissue-restricted patternof transgene expression), as well as quantitative differences in SM22αpromoter activity, the bacterial lacZ gene has been utilized in eachtransgenic construct. Of note, until recently quantitative analysis oftransgene expression has required the use of reporter genes such asfirefly luciferase or bacterial chloramphenicol acetyltransferasebecause of the need for an ultra-sensitive assay that is linear overseveral orders of magnitude. However, an ultra-sensitivechemilluminescent β-galactosidase assay (Tropix) has been developed thathas been used to quantitate β-galactosidase activity in tissues oftransgenic mice. Transgenic constructs will be cloned by isolating themutated SM22α promoter subfragments from the pGL2-Basic luciferasereporter plasmid by digestion with XhoI and HindIII and subcloning thepromoter sequences into the pBS-lacZ transgenic plasmid (apBluescript-based phagemid containing the lacZ gene inserted in thepolylinker). Construction of each plasmid is confirmed by bothrestriction endonuclease mapping and DNA sequence analysis as describedpreviously (Paquet et al., 1990; Parmacek et al., 1990; Parmacek et al.,1994; Parmacek and Leiden, 1989; Parmacek et al., 1992). Transgenic miceare produced as described previously (Metzger et al., 1993). Foundermice are identified by Southern blot analyses as described earlier. Eachrespective construct is represented by at least four founder mice. Ofnote, because transgene expression is affected by both the site ofintegration into the host genome and in some cases by copy number (norelationship between the copy number and β-galactosidase expression hasbeen detected in the four -441 SM22-lacZ lines of transgenic miceanalyzed to date), transgenic offspring from at least four independentfounders with each construct are analyzed.

F0 ED 11.5 transgenic mouse embryos are fixed and histochemicallystained for β-galactosidase activity as described earlier. Whole embryosare stained through ED15.0 (the last day the fixative and stain willpenetrate) and sections of adult mice are stained as describedpreviously (Lin et al., 1990). These analyses determine the function ofeach cis-acting element (SME-1-6) in regulating the spatial and temporalpattern of SM22α gene expression in the developing mouse. To determinethe quantitative effects of each mutation on SM22α promoter function,the normalized β-galactosidase activity of aortic homogenates preparedfrom 6 wk old transgenic mice in which the lacZ gene is under thetranscriptional control of specific SM22α promoter mutants are comparedto the β-galactosidase activity in aortic homogenates prepared from the-441 SM22-lacZ lines of transgenic mice (positive control). Quantitativeβ-galactosidase assays are performed according to the manufacturer'sdirections (Tropix, Inc.) using an LKB/Wallace Bio-orbit Luminometer.β-galactosidase activity is normalized to protein content and expressedas percent of β-galactosidase activity obtained with the wild-typepromoter as described previously (Parmacek et al., 1992). In each study,homogenates from non-transgenic litter mates serve as negative controls.These studies, coupled with earlier results described above, define thefunction of each of the six cis-acting elements within the 441-bp SM22αpromoter both in vitro and in vivo. In addition, these studies addressthe question of whether distinct, common or over-lapping cis-actingelements control transcription of the SM22α gene in arterial SMCs andthe somites during murine embryogenesis. These studies also serve toidentify negative cis-acting regulatory elements that restrict activityof the SM22α promoter to arterial SMCs and the somites by identificationof transgenic mice in which β-galactosidase activity is expressed innon-muscle cell lineages.

EXAMPLE 12 Molecular and Biochemical Analyses of Trans-Acting Factorsthat Regulate SM22α Gene Expression

These studies identify SMC lineage-specific or lineage-restrictedtranscription factors that bind to functionally important nuclearprotein binding sites of the SM22α promoter. Electrophoretic mobilityshift assays (EMSAs) are performed to determine the number andcell-specificity of trans-acting factors that bind to each functionallyimportant nuclear protein binding site in the SM22α promoter. Inaddition, to aid in the functional characterization of each trans-actingfactor, the precise contact residues of each nuclear protein complex andits cognate binding site are identified by methylation and uracilinterference analyses. Finally, to facilitate the cloning and structuralcharacterization of novel SMC-specific transcription factors, thenumber, lineage-specificity, and size of the proteins that compose eachpotentially novel nuclear protein complex are characterized byUV-crosslinking analysis. Of note, nuclear protein complexes that areidentified by EMSAs often represent multi-protein complexes due to thenon-denaturing electrophoresis conditions utilized. Therefore a nuclearprotein complex that appears to be expressed in a ubiquitous fashion mayin fact include lineage-restricted transcription factors.

Electrophoretic Gel Mobility Shift Assays (EMSAs)

The lineage specificity of the nuclear protein complexes that bind toSME-1-6 are analyzed by EMSAs as described (Ip et al., 1994; Parmacek etal., 1994; Parmacek et al., 1992). For these studies, nuclear extractshave been prepared from primary rat aortic SMCs, the rat SMC line, A7r5,murine C2C12 and So18 skeletal myoblasts and myotubes, primary ratcardiac myocytes (90-100% myocytic/10% fibroblasts), mouse NIH 3T3 andC3H10T1/2 cells and human umbilical vein endothelial cells. In addition,double-stranded oligonucleotide probes corresponding to each of thenuclear protein binding sites within the SM22α promoter (see FIG. 3D)have been synthesized and annealed. EMSAs are performed as describedpreviously using both high and low ionic strength binding buffers (Ip etal., 1994; Parmacek et al., 1994; Parmacek et al., 1992). Thespecificity of band shifts (protein-bound DNA) will be determined byperforming cold-competition studies using either unlabeled specific ornon-specific mutated oligonucleotide competitors in the reactionmixture. A band shift that represents a specific DNA-protein interactionis competed only by specific unlabeled oligonucleotides. To identify SMClineage-specific nuclear protein complexes, the binding patternsobtained with SMC (primary rat aortic SMCs and the SMC line A7r5) andnon-SMC extracts are compared. Of note, it is also possible that the SMClineage-specific pattern of SM22α transcription is controlled in whole,or part, by a transcription factor that is expressed exclusively innon-SMCs which binds to the SM22α promoter and suppresses transcription.Therefore, attention is also focused on determining whether a nuclearprotein complex is expressed in non-SMC lineages. Finally, to determinewhether any of the proteins that bind to previously described consensusmotifs located within the SM22α promoter (i.e., SME-2 contains apotential Sp1 binding site and SME-6 contains a potential CREB/ATFbinding site) are antigenically-related, or identical, to knowntranscription factors, antibody supershift reactions are performed asdescribed previously (Johansen and Prywes, 1993). In these studies,pre-immune serum are added to the reaction mixtures that do not containspecific antiserum to control for artifactual differences in bandmigration due to the addition of serum. The mobility of a nuclearprotein complex that contains a protein which is recognized by thespecific antibody will migrate more slowly (be "supershifted") than theDNA-protein complex alone. Alternatively, in some cases the binding of anuclear protein complex which is recognized by the antisera isabolished.

Methylation and Uracil Interference Analyses

To perform methylation interference analyses, 12.5 pM of oligonucleotidecorresponding either to the sense or antisense strands of each of therespective SM22α nuclear protein binding site is phosphorylated with ³²P-γATP and T4 polynucleotide kinase, annealed together and free countsremoved as described previously (Parmacek and Leiden, 1989). 5 ml (5×10⁶DPM) of oligonucleotide is then methylated in 0.05M DMS (whichmethylates guanine residues at the N-7 position and adenines at the N-3position). The methylated probe is purified by sequential precipitationsand EMSAs scaled up 5-10-fold are performed using the radiolabeled,methylated probes and SMC nuclear extracts. The wet gel isautoradiographically exposed overnight and bands corresponding to thecomplexes previously identified by EMSAs, as well as the unbound probe(at the bottom of the gel), are excised and the DNAs electroeluted. Theresuspended DNA pellets are then cleaved with piperidine, lyophilized,and the protein-bound DNA, as well as the unbound free probe areelectrophoretically separated on a 6% DNA sequencing gel. Guanosine (G)residues visible with the free probe and absent in the protein boundprobe represent DNA-protein contact residues. Adenines can also bedetected in this assay but these reactions are much weaker than the Greactions. In all cases, binding to both sense and antisense strands isexamined.

In a complementary series of studies, uracil interference analyses areperformed. To prepare ³² P-labeled deoxyuracil-substituted probes(corresponding to each SM22α nuclear protein binding site), 8-12 cyclesof PCR™ are performed using 0.2 pmol of template DNA, 20 pmol of one(either sense or antisense) [³² P]-end-labeled PCR™ primer, 20 pmol ofthe complementary unlabeled primer (primers are identical to thenucleotide sequences flanking each nuclear protein binding site), 5 μlof 2 mM dNTP mixture, 5 μl of 0.5 mM dUTP, 5 μl of 10×Taq buffer and 1μl of Taq polymerase as described previously (Parmacek and Leiden,1989). The two PCR™ reactions which differ with respect to radiolabeledoligonucleotide primer yield binding site probes that are specific forthe individual strands. The PCR™ products are then purified as describedabove and the primer pairs annealed. Scaled-up EMSAs are then performed,the informative bands (and free probe) isolated, and DNA electroelutedfrom the wet gel slices as described above. The DNAs are then cleaved atthe uracil residues by digestion with uracil-N-glycosylase for 1 h at37° C. The resuspended DNA pellets are then cleaved with piperidine,lyophilized, and electrophoretically separated on a 6% DNA sequencinggel. Thymine residues visible with the free probe and absent in theprotein bound probe represent DNA-protein contact. By sequentiallyidentifying the precise contact residues of each respective nuclearprotein complex identified by EMSAs by both methylation and uracilinterference analyses, a targeted functional assessment of each nuclearprotein complex that binds to each nuclear protein binding sites withinthe SM22α promoter can be undertaken. In addition, DNA-protein bindingconditions may be optimized to selectively isolate and characterize aspecific nuclear protein complex by UV crosslinking and/oroligonucleotide affinity chromatography.

UV-Crosslinking Analysis

Functionally important trans-acting factors that bind to the SM22αpromoter are biochemically characterized by UV-crosslinking toradiolabeled oligonucleotides as described by Chodosh et al. (1986). Inthese studies, a synthetic oligonucleotide (approximately a 40-50 mer)containing a SM22α promoter nuclear protein binding site (as deduced bythe analyses described above) are annealed to a complementary 15-bpsynthetic oligonucleotide derived from the 3' end of this 40-50 bpoligonucleotide. The hybridized complex are radiolabeled using theKlenow fragment of DNA polymerase I in the presence of BrdU and [α³²P]-dCTP. The labeled double-stranded oligonucleotide is purified by PAGEand used in a scaled-up EMSAs containing 105 counts of labeled probe and20 μg of nuclear extract prepared from primary rat aortic SMCs (andnon-SMCs) in the presence of 10 μg of poly-dI:dC. After 15 minutes atroom temperature, the binding reactions is irradiated for periodsbetween 5 and 30 minutes at 305 nm with an intensity of 7,000microwatts/cm². Following irradiation, the reaction mixture will betreated with DNase I and micrococcal nuclease to digest unbound probe.The reaction mixture is then fractionated by SDS-PAGE and the size,number and lineage-specificity of binding proteins determined.Particular emphasis is placed on characterizing SMC-specific nuclearproteins by comparing the DNA binding proteins present in nuclearextracts prepared from SMCs to nuclear proteins present in striatedmuscle and non-muscle cells. SMC lineage-specific trans-acting factorsidentified in these studies, are isolated by either the λgt11 screeningtechnique described by Singh et al. (1988) or byoligonucleotide-affinity chromatography as described by Tijan (Kodonagaand Tijan, 1986).

EXAMPLE 13 Functional Characterization of Transcription Factors thatRegulate the SM22α Promoter in Arterial SMCs

The purpose of these studies is to define the positive or negativeregulatory function of each trans-acting factor on SM22α promoteractivity in SMCs and to examine how combinatorial interactions betweenthese factors regulate SM22α gene expression. To define the function ofa specific trans-acting factor on SM22α promoter function, both loss offunction and gain of function analyses are performed. Of note, becauseCArG elements have been identified in the promoters that controlexpression of several SMC-specific genes and two functionally importantSRF binding sites have been identified in the SM22α promoter (SME-1 andSME-4), particular emphasis is placed on examining the molecularmechanisms underlying the functional activity of the SRF/SME complex inregulating activity of the SM22α. In addition, these studies identifythose trans-acting factors that play important roles in regulating theSMC-specificity of SM22α gene expression. These studies begin to definethe trans-acting factors that direct arterial SMC-specific geneexpression and the molecular program(s) that underlies SMC diversity.

Mutational Analysis of SM22α Promoter

To define the function of specific nuclear protein complexes on SM22αpromoter activity, transient transfection analyses are performed asdescribed earlier. However, in these studies more subtle mutations arecreated in the SM22α promoter that abolish the binding of a specifictrans-acting factor (without affecting binding of other factors). Todefine mutations that abolish binding of a single trans-acting factor, aseries of EMSAs are performed as described above. These mutations areguided by the methylation and uracil interference analyses describedabove. For example, both SRF and YY1 (and two other unrelated nuclearprotein complexes) bind to the SME-4 site in the SM22α promoter. EMSAshave revealed that a two nucleotide substitution in the SME-4 site (5CTCCAACTTGGTGTCTTTCCCCGGATATGGAGCCTGTGTGGAGTG 3', SEQ ID NO: 48, mutatednucleotides are underlined) abolishes SRF binding activity, but does notalter other binding activities. In contrast, a distinct two nucleotidesubstitution (5' CTCCAACTTGGTGTCTTTCCCCAAATTAGGAGCCTGTGTGGAGTG 3', SEQID NO:49) blocks YY1 binding activity, but does not affect any otherbinding activity. To create mutations that selectively abolish bindingof SRF or YY1 to the SME-4 site within the context of the SM22αpromoter, gapped heteroduplex oligonucleotide-mediated or PCR™-mediatedsite directed mutagenesis are performed as described previously(Parmacek et al., 1992). To determine the effect of SRF- versusYY1-binding (at the SME-4 site) on SM22α promoter activity, luciferasereporter plasmids under the transcriptional control of the SRF and YY1SM22α promoter mutants, respectively, are transiently transfected intoprimary cultures of rat aortic SMCs and their activities compared to thenative SM22α promoter containing plasmid, p-441SM22-luc. If, SRF (andSRF accessory factors) binding to SME-4 activates SM22α promoteractivity, the normalized luciferase activity in cells transfected withthe p-441SM22m4/SRF plasmid (which ablates SRF binding at the SME-4site) is less than that obtained with the p-441SM22-luc plasmid.Similarly, if YY1 acts as a positive regulatory factor at the SME-4site, the normalized luciferase activity in SMCs transiently transfectedwith the p-441SM22m4/YY1 plasmid (which ablates binding of YY1 at theSME-4 site) is less than that obtained in cells transfected with thep-441SM22-luc plasmid. Alternatively, if YY1 binding to the SME-4 sitedecreases SM22α promoter activity (i.e., YY1 finctions as a negativeregulatory factor), cells transfected with he p-441SM22m4/YY 1 plasmidhave increased levels of luciferase activity. In addition, multiplecis-acting elements are mutated within the context of the p-441SM22-lucplasmid to determine the function of trans-acting factors that bind tomultiple cis-acting elements within the SM22α promoter. If a nuclearprotein complex is identified that is expressed exclusively in non-SMClineages (potentially a suppressive factor), then the nuclear proteinbinding site that binds this complex is mutagenized within the contextof the p 441SM22luc reporter plasmid and its transcriptional activitycompared to the native 441-bp promoter in non-SMC lines. As it is likelythat several of the nuclear protein complexes identified by EMSA sharecommon binding sites, these studies may only be capable of assigningfunctional activity to one of several nuclear protein complexes.However, elucidation of the precise contact residues between eachnuclear protein complex and its cognate binding site can, in some cases,permit the assignment of functional activity to a specific nuclearprotein complex. Finally, particularly informative promoter mutants areanalyzed in vivo by breeding and histochemically and biochemicallyanalyzing transgenic mice in which the bacterial lacZ is placed underthe transcriptional control of the SM22α promoter mutants as describedearlier.

Examination of the Molecular Mechanisms Underlying the FunctionalActivity of SRF and YY1 on SM22α Promoter Activity

An alternative approach to determine the function of a transcriptionfactor is to over-express a dominant negative form of the transcriptionfactor and examine its effect on activity of a reporter plasmid. Forexample, it has been recently demonstrated that expression of a dominantnegative mutant SRF protein, designated SRF pm1 (Johansen and Prywes,1993), that contains a three amino substitution in the DNA-bindingdomain of SRF, blocks transcriptional activation of the α-skeletal actinand c-fos promoters. (Presumably this mutant protein functions in adominant negative fashion by dimerizing with native SRF inhibiting thenative protein from binding DNA and/or binding critical transcriptionfactors that bind directly to SRF.) To determine whether SRF expressedin arterial SMCs activates transcription of the SM22α promoter, thepCGNpm1 eukaryotic expression plasmid, encoding a flu-epitope tagged SRFpm1 mutant protein (obtained from R. Prywes, Columbia University), andthe p-441SM22-luc reporter plasmid are transiently co-transfected intoprimary rat aortic SMCs and the SMC-line, A7r5, at various molar ratiosas described previously (Parmacek et al., 1994). Expression of thedominant negative protein in SMCs are confirmed by performing westernblot analyses on representative cell lysates using a monoclonal antibodythat recognizes the flu-epitope tag as described previously (Chang etal., 1995). The normalized luciferase activity are compared to thatobtained in cells transfected with the p-441 SM22-luc reporter plasmidand the pcDNA3 negative control expression plasmid. As a positivecontrol, a luciferase reporter plasmid containing the c-fos promoter isco-transfected with and without the pCGNpm1 expression plasmid asdescribed above. Finally, to prove that the dominant negative protein isacting specifically, as opposed to squelching transcription, the pCGNpm1expression plasmid is transiently co-transfected into SMC cultures withseveral non-SRE-dependent promoters. The demonstration that SM22αpromoter activity is down-regulated, or abolished, when a dominantnegative SRF protein is expressed would suggest that SRF is activatingtranscription of the SM22α promoter in arterial SMCs.

The demonstration that the SM22α gene is expressed at high levels inmedial SMCs, but the gene expression is down-regulated to non-detectablelevels in "synthetic SMCs" located within atherosclerotic plaques(Shanahan et al., 1994), suggests that both positive and negativeregulatory mechanisms control expression of the SM22α gene in arterialSMCs. EMSAs (see FIG. 4A and FIG. 4B) revealed that an oligonucleotideprobe corresponding to the SME-4 binds both SRF (a positive regulatoryfactor when activated (Johansen and Prywes, 1995)) and YY1 (which caneither activate or suppress transcription (Natesan and Gilman, 1995a)).In C2C12 skeletal myoblasts, it has been demonstrated that YY1 bindsCArG box sequences (similar to those present in SME-4) in such a waythat it antagonizes SRF action (Gualberto et al., 1992). Moreover,over-expression of YY1 in C2C12 myoblasts has been shown to inhibitdifferentiation of skeletal myoblasts to terminally differentiatedmyotubes (Lee et al., 1992). These data are consistent with thehypothesis that protein-protein and protein-DNA interactions that occurat the SM22α SME-4 nuclear protein binding site serve to activatetranscription by binding transcriptional activators such as SRF (andassociated proteins), or suppress transcription by bindingpreferentially to suppressive factors such as YY1. To test thishypothesis, the pcDNAYY1 expression plasmid, which encodes the mouse YY1protein, is transiently co-transfected with the p-441SM22-luc reporterplasmid into primary rat aortic SMCs and the luciferase activitycompared to that of cells transiently co-transfected with thep-441SM22-luc plasmid and the negative control expression plasmid,pcDNA3 (in the same molar ratios). To determine whether the suppressing(or activating) effect of YY1 is dependent upon its DNA-bindingactivity, the p-441SM22-luc plasmid is transiently co-transfected intoprimary rat aortic SMCs with the pcDNAmYY1 expression plasmid thatencodes a mutant YY1 protein that cannot bind DNA. To determine whetherthe effect of YY1 on SM22α promoter activity is dependent on bindingdirectly to the SM22α promoter (a direct effect versus an indirecteffect), the YY1 expression plasmid is co-transfected with a luciferasereporter plasmid under the transcriptional control of the SM22α promoterwhich has been mutagenized to abolish YY1 binding activity. Finally, todetermine whether YY1-induced suppression of SM22α promoter activity (ifit exists) can be overcome by over-expression of SRF (suggesting adirect antagonism between YY1 and SRF) transient co-transfection studiesis performed as described above except that expression plasmids encodingboth YY1 and SRF are included and their ratios varied over a range ofconcentrations. The demonstration that over-expression of YY1 suppressestranscription from the SM22α promoter would suggest that, as in skeletalmuscle cells, YY1 acts as a negative regulatory factor. Conversely, thedemonstration that over-expression of YY1 increases SM22α promoteractivity would suggest (but not prove) that, as with the c-fos promoter,YY1 acts as a positive regulatory factor (Natesan and Gilman, 1995b).

Transactivation of the Arterial SMC-Specific SM22α Promoter in non-SMCs

To identify transcription factor(s) that play important roles inregulating SMC-specificity of SM22α gene expression, transientco-transfections are performed in non SMC lineages such as NIH3T3 cellsto elucidate the role of GATA-4/5/6 subfamily members in regulatingcardiac-specific transcription as described previously (Ip et al., 1994;Morrisey et al., 1996). Of note, these studies are performed after SMClineage-restricted transcription factor(s) are cloned and structurallycharacterized, or whenever a candidate regulator of the SMC lineage(s)is identified. For example, to determine whether themesodermally-expressed homeodomain transcription factor MHox cantransactivate the SM22α promoter in non-SMCs, NIH 3T3 cells aretransiently transfected with the p-441SM22-luc reporter plasmid and theeukaryotic expression plasmid encoding the full-length MHox protein,pEMSVMHox (obtained from Eric N. Olson), in varying molar ratios (thisratio can vary greatly and must be empirically determined) as describedpreviously (Parmacek et al., 1994). As a negative control, the-441SM22luc plasmid is co-transfected with the pEMSV expression plasmidlacking a cDNA insert and the normalized luciferase activities obtainedfollowing transfection with the pEMSVMHox and pEMSV expression plasmidscompared. To confirm that activation of the SM22α promoter is CArGdependent, the MHox expression plasmid is co-transfected with theplasmid designated p 441SM22mCArG which contains mutations in both theSME-1 and SME-4 nuclear protein binding sites that abolish SRF binding.Next, to determine whether DNA-binding activity of the MHox protein isrequired to activate transcription of the SM22α gene, the cDNA encodingthe (DN144Q)MHox mutant protein which cannot bind DNA (Grueneberg etal., 1992) is subcloned into the pEMSV expression vector and its abilityto transactivate the p-441SM22luc reporter plasmid compared to thepEMSVMHox expression plasmid. If both the native and mutant MHoxexpression plasmids increase transcription of the luciferase reporter toequivalent levels, the ability of MHox (or related homeobox proteins) toactivate transcription of the SM22α promoter does not require directprotein-DNA interaction. Of note, while typically co-transfectionstudies do not require that all of the transcription factors thatactivate a specific lineage-specific promoter be present in therecipient cell line (Ip et al., 1994), it is possible that NIH 3T3 cellsdo not express a specific transcription factor unrelated to SRF that isrequired for SM22α promoter function. In this case, other non-SMC celllines which do not express MHox including COS-1, F9, and P19, are testedin co-transfection studies as described above.

EXAMPLE 14 Cloning and Characterization of Novel SMC-SpecificTranscription Factors that Regulate Activity of the SM22α Promoter

Current developmental paradigms suggest that lineage-specific geneexpression is ultimately controlled by the expression oflineage-specific transcription factors (Olson, 1990; Olson, 1993; Olsonet al., 1991; Olson and Klein, 1994; Tapscott and Weintraub, 1991).Preliminary characterization of these factors have revealed SMC-lineagerestricted factors that bind to SME-3 and non-SRF related nuclearprotein complexes that bind to SME-1 and SME-4 in conjunction with SRF.Novel SMC-specific transcription factors may be cloned using any ofthree complementary approaches. (1) Oligonucleotide affinitychromatography is performed to purify SMC-lineage restrictedtrans-acting factors that regulate activity of the SM22α promoter.

Following purification, these factors are cloned and structurallycharacterized by raising antiserum against the purified protein andusing these antibodies as probes to perform λgt11 library expressionscreening. (2) Alternatively, microsequence analysis of the purifiedprotein fractions are performed followed by oligonucleotidehybridization or PCR™-based library screening based on the partial aminoacid sequence analysis. This approach offers the advantages of isolatingtranscription factors that bind to DNA only as a multi-protein complexesand each step of the protein purification procedure can be evaluatedcritically. However, it has the potential disadvantage of requiringlarge amounts of starting material and its success is to some extentdependent upon the level of protein expressed and the biochemicalproperties of the protein. (3) In a complementary series of studies, aλgt11 mouse aortic cDNA is screened with a radiolabeled oligonucleotideprobe corresponding to a particular SME as described originally by Singhet al. (Singh et al., 1988). While this approach is relativelystraightforward, this cloning approach only detects transcriptionfactors that bind as a single protein or homodimer. Finally, it ispossible that a novel SMC-specific trans-acting factor regulatesactivity of the SM22α promoter via direct protein-protein rather thanprotein-DNA interactions. To identify these potential SMC-specifictranscription factors, two alternative yeast screening selectionstrategies are employed.

Oligonucleotide Affinity Chromatography

Nuclear proteins that bind to the SM22α promoter are isolated bysequence-specific oligonucleotide affinity chromatography as originallydescribed by Tijan and coworkers (Briggs et al., 1986; Kodonaga andTijan, 1986). This approach has successfully been utilized to isolateseveral transcription factors which bind DNA as homodimers, heterodimersor multi-protein complexes (Briggs et al., 1986; Kodonaga and Tijan,1986). The following protein purification strategy is modifiedempirically based on biochemical properties of the isolated protein. Thekey purification step in this strategy is oligonucleotide affinitychromatography which has been demonstrated to result in an 80-foldpurification of protein (Kodonaga and Tijan, 1986). For the sake ofclarity, only a strategy to isolate SME-3 binding factors (a SMC-lineagespecific nuclear protein complex has been identified in EMSAs, see FIG.5B) is presented. However, the protocol described with minor variationsis used to isolate other functionally important lineage-restrictednuclear proteins that bind directly (as determined by EMSAs) to theSM22α promoter.

500 mg-1 g of A7r5 nuclear extract (which contains the lineagerestricted SME-3 binding activity) is prepared as described previously(Parmacek et al., 1992). Ammonium sulfate fractionation are performedand the precipitated protein separated by Sephacryl S-300 gelfiltration. In addition, if necessary additional purification stepsincluding heparin sepharose chromatography and FPLC Mono Q (Pharmacia).Samples containing peak SME-3 binding activity are pooled and subjectedto sequential DNA affinity chromatography as described by Kadonaga andTijan (Kodonaga and Tijan, 1986). An SME-3 DNA affinity resin and amutated SME-3 affinity resin are generated by covalently linkingtandemly ligated copies of the 42-bp double-stranded SME-3oligonucleotide sequence or the mutated SME-3 oligonucleotide sequence,respectively, to cyanogen bromide-activated Sepharose. (Of note, EMSAsand methylation interference analyses are performed in order to definean oligonucleotide sequence (derived from the SME-3 sequence) whichbinds exclusively to the SMC-specific nuclear protein complex ofinterest). This technique results in a matrix containing oligomers ofthe oligonucleotide ranging in size from 3-75 copies. The pooledfractions are mixed with nonspecific competitor DNA, such as calf thymusDNA or poly dI:dC, and applied to the mutated SME-3 affinity resin inorder to remove proteins that non-specifically bind to the SME-3oligonucleotide and column matrix. The column is then washed with 0.1 MKCl and eluted in a multiple step gradient (0.2 M KCl to 1.0 M KCl). Theeluted fraction containing SME-3 binding activity is applied to the wildtype SME-3 affinity resin, washed and eluted as described above. In allof the purification steps, SME-3 binding activity is assayed from columnfractions by performing EMSAs with a radiolabeled SME-3 oligonucleotideprobe. Of note, using this protocol, Tijan and coworkers (Jones et al.,1987; Kodonaga and Tijan, 1986) have demonstrated a cumulative gain inspecific activity of approximately 30,000-50,000-fold/mg protein and acumulative yield of approximately 0.002%. Thus, if one starts with 1 gof nuclear extract, one would expect approximately 20 mg of purifiedprotein utilizing this strategy. If successful (i.e., purification togreater than 95% homogeneity), this amount of protein is sufficient formicrosequence analysis and/or sufficient to raise a specific antiseradirected against the putative SME-3-binding protein(s).

The recovery of SME-3 binding activity and the relative extent ofpurification is analyzed by SDS-PAGE and EMSAs. Following affinitychromatography, protein fractions which bind DNA are visualized bySDS-PAGE and silver staining as described previously (Samarel et al.,1987). To demonstrate which of the polypeptide species isolated by DNAaffinity chromatography and visualized by PAGE represent SME-3 bindingactivity, approximately 2-3 mg of purified protein are separated bypreparative SDS-PAGE and the region of the gel containing the majorpolypeptide species is excised and eluted from the polyacrylamide gel in50 mM Tris, pH 7.9, 0.1 mM EDTA, 0.1% SDS, 5 mM DTT, and 150 mM NaCl.SDS is removed by acetone precipitation, and the precipitated protein isresuspended in TM buffer (50 mM Tris, pH 7.9, 12.5 mM MgCl2, 1 mM EDTA,1 mM DTT, 20% glycerol) containing 0.1 M KCl, 0.1% NP40, and 6Mguanidine HCl to denature the polypeptide chains as described previously(Kodonaga and Tijan, 1986). The guanidine HCl is removed by gelfiltration to facilitate refolding of the polypeptide chains andrecovery of DNA binding activities as determined by EMSA and DNase Ifootprinting is assessed. Of note, the net yield after these two stepsis approximately 20% (Briggs et al., 1986; Jones et al., 1987; Kodonagaand Tijan, 1986). In the EMSAs, a control sample containing excised andrenatured proteins from other regions of the gel, and also non-renaturedprotein are run as controls. Of note, several eukaryotic transcriptionfactors including Sp1 (Briggs et al., 1986), AP-1 (Kodonaga and Tijan,1986), and bacterial transcription factors including sigma factors(Helmann and Chamberlin, 1988) have been functionally reconstitutedusing this technique.

To more precisely characterize the SME-3 binding protein(s), theindividual proteins of the SME-3 complex are purified as describedabove, and undergo amino acid sequence analysis. Tryptic peptides aregenerated, resolved by reverse phase HPLC, and sequenced using anApplied Biosystems gas phase sequencer. These sequences are comparedagainst the GenBank and NBRF data basis to determine if they correspondto previously described proteins or genes. In addition, polyclonalrabbit anti-SME-3 antiserum are prepared as described previously (Ip etal., 1994). Antiserum is screened for activity by western blotting andimmunoprecipitation of A7r5 nuclear extracts as described above. If thisis not successful, synthetic peptides derived from the microsequenceanalyses of the SME-3 binding proteins are used to immunize rabbits.

The sizes of the proteins identified by these western blotting andimmunoprecipitation approaches are compared to those determined in theUV-crosslinking studies described above. In a complementary set ofstudies, it is determined if the antisera recognize the SME-3 bindingactivities by EMSA as described above. The demonstration that theantiserum raised against the SME-3-binding protein is able tospecifically shift SME-3 nuclear protein complexes and pre-clear SME-3binding activity from SMC extracts is strong evidence in favor of theauthenticity of the SME-3-binding protein. Finally, to fullycharacterize each DNA binding protein, oligo-dT primed and random primedmouse aortic λgt11 cDNA libraries are screened, using the antiseraraised against the SME-3 binding protein as described (Gottesdiener etal., 1988). In addition, PCR™-based screening of these libraries isperformed with redundant synthetic oligonucleotide primers derived fromthe amino acid sequence analysis of the tryptic peptide fragments whichare generated from the SME-3 microsequence analysis performed above asdescribed (Wilkie and Simon, 1991).

λgt11 Library Screen

To clone cDNAs encoding proteins that bind to functionally importantnuclear protein binding sites within the SM22α promoter, a large (8×10⁶recombinants) randomly primed λgt11 cDNA library has been constructedusing poly A⁺ RNA prepared from the aorta of 6 wk. old Balb/C mice. Thislibrary is screened for functionally important SMC-specific SM22αpromoter binding activities using a radiolabeled multimericoligonucleotide probe corresponding to the nucleotide sequence of theSME to which novel SMC-specific trans-acting factors bind using amodification of the procedure of Singh et al. (Singh et al., 1988) andStaudt et al. (Staudt et al., 1988). Of note, because nucleotidesflanking CArG/SRE elements are often required to isolate factors thatbind in conjunction with SRF (Johansen and Prywes, 1995), in order toisolate factors that bind to SME-1 or SME-4 the oligonucleotide probesutilized include 25-bp of 5' and 3' sequence flanking each embedded CArGmotif. To isolate the novel SMC lineage-restricted SME-3 bindingfactors, a radiolabeled SME-3 probe is prepared by annealing of singlestranded SME-3 oligonucleotides followed by ligation of overhangingBamHI and BglII ends and nick translation with [α³² P]-dCTP. 1×10⁶ phagefrom the library are plated, incubated for approximately 3.5 h at 41°C., overlaid with nitrocellulose filters that have been presaturatedwith 10 mM IPTG, and incubated an additional 3.5 h at 37° C. Thefilter-bound proteins are then denatured and renatured through guanidineHCl and HEPES binding buffers (this step is optional) and blocked withBlotto. Filter hybridization is performed for 1 h at 25° C. in asolution

containing 2×10⁶ DPM/ml of radiolabeled probe, TNE 50 (10 mM Tris, pH7.5, 50 mM NaCl, 1 mM EDTA and 1 mM DTT) and denatured calf thymus DNA.Following hybridization, filters are washed in TNE 50, dried andautoradiography is performed to identify positively hybridizing clones.The specificity of binding is tested by hybridizing tertiary filters toradiolabeled SME-3 and mutant SME-3 (mutations which inhibited bindingare confirmed by EMSAs), as well as, unrelated oligonucleotide probes.Only cDNA that bind to the specific oligonucleotide probe, but not tothe mutant and non-specific probes are characterized further.Subsequently, each candidate SME-3 clone is purified to homogeneity bysequential screening with the same radiolabeled probes and the insertssubcloned into pGEM4Z (Promega).

To assess the binding specificity of the proteins encoded by these SME-3candidate clones, lambda lysogens are made by infecting E. coli Y1089with the SME-3 λgt11 candidate clones at 32° C. Fusion proteinexpression is induced by temperature shift to 44° C. and IPTG treatmentand crude bacterial lysates are prepared as per Singh (Singh et al.,1988). Control lysates are identically prepared from Y1089 lysogenscontaining wild type λgt11. The SME-3 lysates are tested for SME-3binding activities by EMSAs (including cold competition studies andEMSAs with mutant SME-3 oligonucleotide) as previously described. Inaddition, the lysates are subjected to SDS-PAGE and duplicate westernblots are screened with either radiolabeled SME-3 oligonucleotide probe(as well as non-specific negative control probe) and with commerciallyavailable anti β-galactosidase antibody (Capel).

If the cDNA clone(s) are shown to encode proteins which bindspecifically to the SME-3 oligonucleotide, two types of studies areperformed to attempt to determine whether they encode the SME-3 bindingactivities present in SMC nuclear extracts. First, rabbit antisera israised against the recombinant SME-3 fusion protein and to show thatthese antisera are both able to recognize SMC nuclear proteins of thesame size as those identified in the UV-crosslinking studies describedabove, and to specifically block the SME-3 binding activities in SMCnuclear extracts. Briefly, SME-3 fusion proteins are prepared from crudelysates of λgt11 lysogens by preparative SDS-PAGE (following stainingwith 0.1 M KCl) and bands cut from the gels, crushed, and mixed withadjuvant. 5-10 mg of this gel purified fusion protein (in crushedacrylamide) are injected into female rabbits subcutaneously at 2-3 weekintervals. Seven days following immunization, serum is collected andextensively preabsorbed by incubation with extracts of wild type λgt11lysogenized Y1089 in order to remove anti-β-galactosidase andanti-bacterial protein activities. Pre-immune and immune sera are usedto probe duplicate western blots containing SDS-PAGE fractionatedproteins from the SME-3 lysogens as well as wild type λgt11 lysogens. Ifspecific antisera are not obtained following immunization with fusionproteins, synthetic immunogenic peptides designed from DNA sequenceanalysis of the cDNA clone(s) using the Hopp and Woods algorithm of theWisconsin GCG software package (Madison, Wis.) are used to immunizerabbits and the resulting antisera assayed by western blotting asdescribed above. Sera which recognize the SME-3 fusion protein, but notthe wild type λgt11 proteins are then assayed for their ability torecognize SME-3 protein from SMCs by western blotting of SMC extractsand by immunoprecipitation of ³⁵ S-methionine labeled SMC nuclearextracts as previously described (Martin et al., 1993). The sizes of theproteins identified by these western blotting and immunoprecipitationapproaches are compared to those determined in the UV-crosslinkingstudies described above. In a complementary set of studies, an attemptto determine whether the antisera recognize the SME-3 binding activityis made by EMSA. In these studies, the radiolabeled SME-3oligonucleotide is incubated with SMC nuclear extract either in thepresence of control (pre-immune) or specific antisera and the resultingcomplexes assayed by standard EMSA. If the antiserum recognizes one ofthe nuclear proteins, it should result in a specific further shiftupwards in mobility in the gel ("a supershift"). The antisera are alsoused to preclear SMC nuclear extracts by immunoprecipitation. Thesepre-cleared extracts are then assayed for SME-3 binding activities bygel mobility shift assays using radiolabeled SME-3 probe. Thespecificity of preclearing is confirmed by simultaneously assaying theprecleared nuclear extracts of another SME by EMSA. The demonstrationthat the antiserum raised against the recombinant fusion protein is ableto specifically shift and preclear SME-3 binding activities from SMCextracts would be strong evidence in favor of the authenticity of theSME-3 candidate cDNA clones.

In a second set of studies, the SME-3 cDNAs are used as hybridizationprobes on northern blots containing RNA from neonatal and adult murinetissues as well as primary rat aortic SMCs (which contain less than 5%contaminating fibroblasts) in order to determine the number and sizes ofmRNA species corresponding to these cDNAs. If this cDNA is a bona fideSMC-specific transcription factor which is regulated at the level ofgene expression, it is expected that it would be expressed at highlevels in SMC-enriched tissues such as the aorta and in primary rataortic SMCs (it is extremely likely that the mouse and rat cDNA clonescross-hybridize). However, it is also possible that SME-3 candidate cDNArepresents a ubiquitously expressed transcription factor which binds tothe SME-3 binding site, or that the bona fide gene which encodes SME-3mRNA respectively is expressed in multiple cell lineages and itsactivity in SMCs is regulated at the translational or post-translationallevel. Thus, the finding that SME-3 mRNA is not restricted to SMCs doesnot rule out its authenticity as a bona fide SME-3 binding factor whichmay bind specifically in vascular SMCs. Once the SME-3 cDNA clone(s) areauthenticated in the studies described above, full length clones for DNAsequence analysis are isolated. Full length clones are either obtaineddirectly or assembled from overlapping cDNAs obtained by screening thelarge oligo-dT primed and random hexamer-primed murine aortic λgt11libraries with probes from the 5' and 3' ends of the SME-3 cDNA. cDNAsare subcloned into pGEM4Z and sequenced directly from double strandedplasmid DNA as described previously (Solway et al., 1995). cDNAsequences are analyzed using the MacVector software package (Oxford) andthe GenBank and NBRF databases are searched in order to detectsimilarities with previously described proteins and genes.

If these approaches are unsuccessful in identifying SME-3 cDNA clones,an alternative cDNA expression cloning approach can be used. A largerandomly primed cDNA library is constructed with poly A+ mouse aorta RNAin the commercially available pCDM8 vector. In this vector,transcription of cDNAs is under the control of a CMV promoter. Inaddition, the vector contains an SV40 origin of replication to allowhigh copy amplification in COS cells. Pools of 5,000 clones of this cDNAlibrary are transfected into COS cells and nuclear extracts are preparedfrom the transfected cells after 72 h. These nuclear extracts along withcontrol extracts from mock-transfected COS cells are assayed for SME-3binding activity by EMSAs. If one or more pools are found to encode theSME-3 binding activity, cDNA clones encoding SME-3 are isolated bysib-selection using EMSAs. Of note, this approach was used by Orkin andco-workers to isolate the erythroid specific transcription factor, NF-E2(Orkin, 1992). The success of this technique depends upon theuntransfected COS cell nuclear extracts not displaying this potentiallynovel SME-3 binding activity.

Yeast Two Hybrid Screen

A number of studies suggest that interactions between MADS boxtranscription factors and lineage-restricted accessory proteins couldestablish cell identity, in part, by determining which genes areactivated in response to generic inductive signals which are transducedby (Marais et al., 1993; Mueller and Nordheim, 1991; Pellegrini et al.,1995) ubiquitously-expressed transcription factors such as SRF (Cserjesiet al., 1994; Grueneberg et al., 1992). As discussed above, the SM22αpromoter contains tandem binding sites for the MADS box transcriptionfactor SRF (SME-1 and SME-4) suggesting that protein-proteininteractions between SRF and other accessory factors expressed in SMCscould regulate SMC-specific transcription. One approach that has beensuccessfully utilized to isolate cDNA clones that bind directly to atranscription factor is to perform a genetic screen in yeast (Fields andSong, 1989). Of note, an advantage of this strategy is that it is basedon in vivo interactions between proteins when they are expressed intheir native conformation. However, an inherent limitation of the yeasttwo hybrid screening strategy is that it is dependent upon functionalcomplementation by a single cDNA (or deduced protein). Despite thislimitation, similar strategies have successfully identified manyfunctionally important protein partners (Dalton and Treisman, 1992;Grueneberg et al., 1992). Moreover, as described above, the alternative(and complementary) strategy of oligonucleotide affinity chromatography(which is not bound by this potential limitation), are also performed.

Initially, a modification of the yeast screening selection strategyemployed by Dalton and Treisman to isolate SAP-1 from a HeLa cell cDNAlibrary (Dalton and Treisman, 1992) is used. This strategy offers theadvantage of isolating proteins that require both protein-proteininteraction (with SRF) and specific protein-DNA binding (to the SME-1 orSME-4) to activate transcription. Because the yeast MADS boxtranscription factor MCM1 binds to some CArG/SRE elements (which wouldpreclude their use in the library screen described below), it is firstdetermined whether MCM1 binds to the SM22α SME-4 (or SME-1) site by EMSAand by two hybrid complementation studies in yeast as described (Nurrishand Treisman, 1995). Of, note 9 out of 10 nucleotides in the SME-4CArG/SRE motif (5' CCAAATATGG 3', SEQ ID NO:50) are identical to thenucleotides that compose the skeletal α-actin CArG box (5' CCATATATGG,SEQ ID NO:51) which binds exclusively to SRF (and not to MCM1) (Nurrishand Treisman, 1995). To prepare the yeast indicator strain, doublestranded oligonucleotides corresponding to the SME-4 nuclear proteinbinding site and 25-bp flanking the embedded CArG/SRE motif aresubcloned into the XhoI site 5' of the CYC1 TATA box in the CYC1/LacZreporter plasmid, pLGD-178 (Guarente et al., 1982). The SME-4/lacZreporter gene is excised as an XhoI-NcoI fragment and inserted into theStuI site of pURA3 (Dalton and Treisman, 1992), thereby embedding thereporter gene in the S. cerevisiae URA3⁺ coding region. Each URA3disruption plasmid is digested with HindIII to release the URA3 reportersequences and is cotransformed with a TRP1⁺ marker plasmid (pRS314) intothe URA3⁺ S. cerevisiae strain S50 (HMLα, MATα, HMRa , his3-11, 15,trp1-1, ade2-1, leu2-3, 112, URA3⁺ ho, can1-100) by spheroplasttransformation as described (Hinnen et al., 1978). Several hundred Trp⁺transformants are pooled, resuspended in minimal medium (minus aminoacids) and selected for resistance to 5-fluorotic acid (5-FOA, Sigma).Individual colonies are picked from the 5-FOA plates, re-purified,streaked on ura- selective plates, and checked by Southern blot analysisto confirm disruption of the URA3⁺ locus by the reporter gene. Next, thelow copy plasmid, pSD.07 (Dalton and Treisman, 1992), that permitsgalactose-inducible expression of SRF and contains the TRP1+ marker aretransformed into the Y.SME-4 cells creating the Y.SME-4/SD.07 indicatorstrain.

For these studies, a VP16-tagged rat aortic SMC cDNA library is preparedby cloning randomly primed mouse aortic cDNA into the BstX1 site of theyeast expression vector pSD.10 (this vector permits galactose-inducibleexpression of the fusion protein) as described previously (Solway etal., 1995). A culture of Y.SME-4/SD.07 is transformed with the VP16-tagged cDNA library, plated on nylon filters and grown on ura- trp-selective glucose medium for 32-38 hours at 30° C. Filters are thentransferred to galactose plates for a further 18 h to induce expressionof both the cDNA library and SRF. A colony color β-galactosidaseactivity assay is then performed as described by Breeden and Nasmyth toidentify lacZ-positive (blue) colonies (Breeden and Nasmyth, 1987). Bluecolonies are patched on selective medium containing 2% glucose, purified(by growth in ura- trp- selective broth) and plated. After 48 h at 30°C., single colonies are replica plated onto nylon filters and retestedfor colony color. To test whether the positive colonies require thepresence of the VP16/cDNA fusion protein or SRF, cells are cured of theURA1+ cDNA (which is carried on the pSD.10 vector) or TRP1+ (which iscarried on the SRF expression plasmid) plasmids and retested forβ-galactosidase activity. Of note, this will serve to distinguish SMCcDNA clones that activate the reporter plasmid in the absence of SRF(approximately 25% of clones (Dalton and Treisman, 1992)) which is apotential artifact of the two-hybrid strategy. VP16/cDNA plasmids arethen recovered from strains cured of the TRP1+ plasmid (SRF expressionplasmid), transformed into bacteria, isolated and characterized asdescribed.

Alternatively, a standard yeast two hybrid screen is performed. Of note,in contrast to the screening strategy described above, this strategyonly isolates those trans-acting factors that bind to SRF in anon-DNA-dependent fashion. In these studies the yeast indicator strainHF7c (ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112,gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::GAL4-lacZ) (Clontech) whichcarries the GAL1-HIS3 and GAL4-lacZ reporters and the trp1, leu2transformation markers are utilized. This strategy offers the potentialadvantage of utilizing "bait plasmids" that encode specific domains ofthe SRF protein (as opposed to the full-length SRF protein) (Johansenand Prywes, 1993) thereby decreasing background from proteins thatinteract non-specifically with SRF. SRF domains that are subcloned intothe pGBT9 plasmid (Clontech) include cDNAs encoding amino acids 168-222(the TCF interaction domain), amino acids 1-406 (the entire SRF proteinexcept the C-terminal activation domain), amino acids 406-476 (theC-terminal activation domain) and amino acids 1-508 (the entire SRFprotein). Of note, if expression of these proteins is "toxic" to theyeast, or if the SRF activation domain activates transcription from theyeast reporter gene, bait plasmids containing specific mutations in theDNA-binding, homodimerization and transcription activation domains aretested (Johansen and Prywes, 1993; Johansen and Prywes, 1994). A GAL4activation domain (AD)/mouse aortic cDNA fusion library is utilized thatis cloned into the pGAD424 vector (Clontech). HF7c cells areco-transformed with the pGBT9 bait plasmid and the pGAD424 GAL4AD/mouseaortic cDNA expression library as described above and the transformedcultures plated on medium minus tryptamine and leucine to select forcells having both plasmids. In addition, because the HF7c indicatorstrain is auxotrophic for histidine, but carries a HIS3 gene under thecontrol of the GAL1 UAS, transformation on medium minus histidineselects for plasmids that interact. A β-galactosidase filter assay isthen performed as described above to confirm the presence of interactingproteins.

Finally, full-length cDNA clones encoding potentially novel SRFaccessory proteins expressed in SMCs are isolated by screening a randomprimed mouse aortic λgt11 cDNA library using the radiolabeled SMC cDNA(isolated by the yeast screening strategies described) as a probe asdescribed previously (Parmacek and Leiden, 1989; Solway et al., 1995).To determine if the putative accessory protein and SRF interact inmammalian cells, NIH 3T3 cells are transiently co-transfected with aluciferase reporter plasmid, pSME-4TKluc, that contains a multimerizedSME-4 oligonucleotide positioned 5' of the minimal HSV TK promoterdriving expression of the firefly luciferase gene (the p-441 SM22lucplasmid which contains two CArG elements is also tested) and anexpression plasmid, designated pVP16SMCDNA3, containing the VP16activation domain fused in-frame to the open reading frame of the novelcDNA in the pcDNA3 eukaryotic expression plasmid. Of note, SRF isnormally expressed in NIH 3T3 cells, yet the minimal SRE-linked HSV TKpromoter is relatively inactive in these cells (Dalton and Treisman,1992). Therefore, if the accessory protein and SRF functionallysynergize in mammalian cells, co-transfection of the CArG-dependentluciferase reporter plasmid and the cDNA expression plasmid shouldincrease transcription of the luciferase reporter gene above levelsobtained following co-transfection of pSRETKluc and an unrelatedVP16-linked cDNA expression plasmid. To determine whether activationrequires DNA binding by SRF, co-transfection is repeated using aluciferase reporter plasmid that contains mutations in the multimerizedCArG motifs. Finally, to examine the molecular mechanisms underlying thefunctional activity of the SMC accessory factor, EMSAs are performedusing the in vitro translated protein isolate and SRF at differentratios. Taken together, these studies should identify and functionallycharacterize each transcription factor(s) expressed in SMCs thatregulates activity of the SRF/SME complex. Moreover, these studies maydirectly isolate novel SMC-lineage restricted transcription factors thatfunction in concert with SRF in regulating transcription of the SM22αgene.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 55                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1419 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GAATTCAGGA CGTAATCAGT GGCTGGAAAG CAAGAGCTCT AGAGGAGCTC CA - #GCTTATTA         60                                                                          - TGACCCTTCC TTCAGATGCC ACAAGGAGGT GCTGGAGTTC TATGCACCAA TA - #GCTTAAAC        120                                                                          - CAGCCAGGCT GGCTGTAGTG GATTGAGCGT CTGAGGCTGC ACCTCTCTGG CC - #TGCAGCCA        180                                                                          - GTTCTGGGTG AGACTGACCC TGCCTGAGGG TTCTCTCCTT CCCTCTCTCT AC - #TCCTTTCT        240                                                                          - CCCTCTCCCT CTCCCTCTCT CTGTTTCCTG AGGTTTCCAG GATTGGGGAT GG - #GACTCAGA        300                                                                          - GACACCACTA AAGCCTTACC TTTTAAGAAG TTGCATTCAG TGAGTGTGTG AG - #ACATAGCA        360                                                                          - CAGATAGGGG CAGAGGAGAG CTGGTTCTGT CTCCACTGTG TTTGGTCTTG GG - #TACTGAAC        420                                                                          - TCAGACCATC AGGTGTGATA GCAGTTGTCT TTAACCCTAA CCCTGAGCCT GT - #CTCACCTG        480                                                                          - TCCCTTCCCA AGACCACTGA AGCTAGGTGC AAGATAAGTG GGGACCCTTT CT - #GAGGTGGT        540                                                                          - AGGATCTTTC ACGATAAGGA CTATTTTGAA GGGAGGGAGG GTGACACTGT CC - #TAGTCCTC        600                                                                          - TTACCCTAGT GTCTCCAGCC TTGCCAGGCC TTAAACATCC GCCCATTGTC AC - #CGCTCTAG        660                                                                          - AAGGGGCCAG GGTTGACTTG CTGCTAAACA AGGCACTCCC TAGAGAAGCA CC - #CGCTAGAA        720                                                                          - GCATACCATA CCTGTGGGCA GGATGACCCA TGTTCTGCCA CGCACTTGGT AG - #CCTTGGAA        780                                                                          - AGGCCACTTT GAACCTCAAT TTTCTCAACT GTTAAATGGG GTGGTAACTG CT - #ATCTCATA        840                                                                          - ATAAAGGGGA ACGTGAAAGG AAGGCGTTTG CATAGTGCCT GGTTGTGCAG CC - #AGGCTGCA        900                                                                          - GTCAAGACTA GTTCCCACCA ACTCGATTTT AAAGCCTTGC AAGAAGGTGG CT - #TGTTTGTC        960                                                                          - CCTTGCAGGT TCCTTTGTCG GGCCAAACTC TAGAATGCCT CCCCCTTTCT TT - #CTCATTGA       1020                                                                          - AGAGCAGACC CAAGTCCGGG TAACAAGGAA GGGTTTCAGG GTCCTGCCCA TA - #AAAGGTTT       1080                                                                          - TTCCCGGCCG CCCTCAGCAC CGCCCCGCCC CGACCCCCGC AGCATCTCCA AA - #GCATGCAG       1140                                                                          - AGAATGTCTC CGGCTGCCCC CGACAGACTG CTCCAACTTG GTGTCTTTCC CC - #AAATATGG       1200                                                                          - AGCCTGTGTG GAGTGAGTGG GGCGGCCCGG GGTGGTGAGC CAAGCAGACT TC - #CATGGGCA       1260                                                                          - GGGAGGGGCG CCAGCGGACG GCAGAGGGGT GACATCACTG CCTAGGCGGC CT - #TTAAACCC       1320                                                                          - CTCACCCAGC CGGCGCCCCA GCCCGTCTGC CCCAGCCCAG ACACCGAAGC TA - #CTCTCCTT       1380                                                                          #  1419            CAAG CCTTGTAAGT GCAAGTCAT                                  - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 991 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 38..218                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 322..500                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 866..967                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #AAG GGT CCA      55TAC TTTAGCTCTG CCTCAAC ATG GCC AAC                        #     Met Ala Asn Lys Gly Pro                                                 #    5  1                                                                     - TCC TAC GGC ATG AGC CGA GAA GTG CAG TCC AA - #A ATT GAG AAG AAG TAT          103                                                                          Ser Tyr Gly Met Ser Arg Glu Val Gln Ser Ly - #s Ile Glu Lys Lys Tyr           #             20                                                              - GAC GAG GAG CTG GAG GAG CGA CTA GTG GAG TG - #G ATT GTA GTG CAG TGT          151                                                                          Asp Glu Glu Leu Glu Glu Arg Leu Val Glu Tr - #p Ile Val Val Gln Cys           #         35                                                                  - GGC CCT GAT GTA GGC CGC CCA GAT CGT GGG CG - #C CTG GGC TTC CAG GTG          199                                                                          Gly Pro Asp Val Gly Arg Pro Asp Arg Gly Ar - #g Leu Gly Phe Gln Val           #     50                                                                      #AATCTAGGGA           248 G TGAGTAACCC TTGCGAAGGG                             Trp Leu Lys Asn Gly Val                                                       # 60                                                                          - TGTGTATGCC GCCCTACAAA CTGTGAGACA GACTCCCTGA GCTGAGTGTT CA - #GTTGTGTT        308                                                                          - CTGTACCTGG CAG ATT CTG AGC AAA TTG GTG AAC AG - #C CTG TAT CCT GAG           357                                                                          #Ser Lys Leu Val Asn Ser Leu Tyr Pro Glu                                      #              10                                                             - GGA TCG AAG CCA GTG AAG GTG CCT GAG AAC CC - #A CCC TCC ATG GTC TTT          405                                                                          Gly Ser Lys Pro Val Lys Val Pro Glu Asn Pr - #o Pro Ser Met Val Phe           #         25                                                                  - AAG CAG ATG GAA CAG GTG GCT CAA TTC TTG AA - #G GCA GCT GAA GAT TAT          453                                                                          Lys Gln Met Glu Gln Val Ala Gln Phe Leu Ly - #s Ala Ala Glu Asp Tyr           #     40                                                                      - GGA GTC ATC AAG ACT GAC ATG TTC CAG ACT GT - #T GAC CTC TAT GAA GG           500                                                                          Gly Val Ile Lys Thr Asp Met Phe Gln Thr Va - #l Asp Leu Tyr Glu               # 55                                                                          - TATAAGGAAA AAAGGGCTGG AGCCAGTGGG CGAGTGGAGA GCAAGATTAT CA - #GTCAAGGA        560                                                                          - GAAGGAATAT CAAAAGCCAC AACCAGCTCT GTTGATGTGT TCATAGCAGG AA - #TGGGATAT        620                                                                          - GCCAAGAGAA CACATAGCAA GGGGACCAGC TTGGTGGTAC AGCATTTCCT TC - #TGGGTACA        680                                                                          - AGGGCCTGTT TTGGATCCTA GAATATCAAA TATATACCAC ACCATACTCA CT - #AGGGTTTA        740                                                                          - GAATATGGTC TCTTGAACCC TCTTGATTTG GTGCCACTTG CTCCTTGGTT GG - #ACCATTTT        800                                                                          - TGAAGCTGGG CAGGTATTGC CTATATGGTC CTGAAATTAG CTCCCTGGCC AC - #TCTTCTCA        860                                                                          #CTA ATG GCT TTG GGC        907 CAG AGG ACT                                   #Arg Thr Leu Met Ala Leu Glyl Gln                                             #     10                                                                      - AGT TTG GCT GTG ACC AAA AAC GAT GGA AAC TA - #C CGT GGA GAT CCC AAC          955                                                                          Ser Leu Ala Val Thr Lys Asn Asp Gly Asn Ty - #r Arg Gly Asp Pro Asn           # 30                                                                          #      991         ATGTGTCCA CTGGGTCTCT CTGT                                  Trp Phe Met Lys                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 60 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Ala Asn Lys Gly Pro Ser Tyr Gly Met Se - #r Arg Glu Val Gln Ser         #                 15                                                          - Lys Ile Glu Lys Lys Tyr Asp Glu Glu Leu Gl - #u Glu Arg Leu Val Glu         #             30                                                              - Trp Ile Val Val Gln Cys Gly Pro Asp Val Gl - #y Arg Pro Asp Arg Gly         #         45                                                                  - Arg Leu Gly Phe Gln Val Trp Leu Lys Asn Gl - #y Val                         #     60                                                                      - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 59 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ile Leu Ser Lys Leu Val Asn Ser Leu Tyr Pr - #o Glu Gly Ser Lys Pro         #                 15                                                          - Val Lys Val Pro Glu Asn Pro Pro Ser Met Va - #l Phe Lys Gln Met Glu         #             30                                                              - Gln Val Ala Gln Phe Leu Lys Ala Ala Glu As - #p Tyr Gly Val Ile Lys         #         45                                                                  - Thr Asp Met Phe Gln Thr Val Asp Leu Tyr Gl - #u                             #     55                                                                      - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 34 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Lys Asp Met Ala Ala Val Gln Arg Thr Leu Me - #t Ala Leu Gly Ser Leu         #                 15                                                          - Ala Val Thr Lys Asn Asp Gly Asn Tyr Arg Gl - #y Asp Pro Asn Trp Phe         #             30                                                              - Met Lys                                                                     - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 575 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..169                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #AAG AGG GAC         51 TTCTAGG AAA GCC CAG GAG CAT                           #            Lys Ala Gln Gl - #u His Lys Arg Asp                              #           5  1                                                              - TTC ACA GAC AGC CAA CTG CAG GAG GGG AAG CA - #C GTC ATT GGC CTT CAA           99                                                                          Phe Thr Asp Ser Gln Leu Gln Glu Gly Lys Hi - #s Val Ile Gly Leu Gln           #     20                                                                      - ATG GGC AGC AAC AGA GGA GCC TCG CAG GCT GG - #C ATG ACA GGC TAT GGG          147                                                                          Met Gly Ser Asn Arg Gly Ala Ser Gln Ala Gl - #y Met Thr Gly Tyr Gly           # 40                                                                          - CGA CCC CGG CAG ATC ATC AGT T AGAAAGGGAA GGCC - #AGCCCT GAGCTGCAGC           199                                                                          Arg Pro Arg Gln Ile Ile Ser                                                                    45                                                           - ATCCTGCTTA GCCTGCCTCA CAAATGCCTA TGTAGGTTCT TAGCCCTGAC AG - #CTCTGAGG        259                                                                          - TGTCACTGGG CAAAGATGAC TGCACATGGG CAGCTCCCAC CTATCCTTAG CC - #TCAGCCCA        319                                                                          - GCATCTTACC CCAGAGCCAC CACTGCCCTG GCCCCTGTTC CCAGCTGTAC CC - #CCACCTCT        379                                                                          - ACTGTTCCTC TCATCCTGGA GTAAGCAGGG AGAAGTGGGC TGGGGTAGCT GG - #CTGTAGGC        439                                                                          - CAGCCCACTG TCCTTGATAT CGAATGTCCT TTGAAGGAGA CCCAGCCCAG CC - #TCTACATC        499                                                                          - TTTTCCTGGA ATATGTTTTT GGGTTGAAAT TCAAAAAGGA AAAAAGAAAA AT - #ATATAAAT        559                                                                          #   575                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Lys Ala Gln Glu His Lys Arg Asp Phe Thr As - #p Ser Gln Leu Gln Glu         #                 15                                                          - Gly Lys His Val Ile Gly Leu Gln Met Gly Se - #r Asn Arg Gly Ala Ser         #             30                                                              - Gln Ala Gly Met Thr Gly Tyr Gly Arg Pro Ar - #g Gln Ile Ile Ser             #         45                                                                  - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1102 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 77..681                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - GCCCGTCTGC CCCAGCCCAG ACACCGAAGC TACTCTCCTT CCAGTCCACA AA - #CGACCAAG         60                                                                          #TAC GGC ATG AGC        109 AAC AAG GGT CCA TCC                               #Met Ala Asn Lys Gly Pro Ser Tyr Gly Met S - #er                              #                 10                                                          - CGA GAA GTG CAG TCC AAA ATT GAG AAG AAG TA - #T GAC GAG GAG CTG GAG          157                                                                          Arg Glu Val Gln Ser Lys Ile Glu Lys Lys Ty - #r Asp Glu Glu Leu Glu           #             25                                                              - GAG CGA CTA GTG GAG TGG ATT GTA GTG CAG TG - #T GGC CCT GAT GTA GGC          205                                                                          Glu Arg Leu Val Glu Trp Ile Val Val Gln Cy - #s Gly Pro Asp Val Gly           #         40                                                                  - CGC CCA GAT CGT GGG CGC CTG GGC TTC CAG GT - #G TGG CTG AAG AAT GGT          253                                                                          Arg Pro Asp Arg Gly Arg Leu Gly Phe Gln Va - #l Trp Leu Lys Asn Gly           #     55                                                                      - GTG ATT CTG AGC AAA TTG GTG AAC AGC CTG TA - #T CCT GAG GGA TCG AAG          301                                                                          Val Ile Leu Ser Lys Leu Val Asn Ser Leu Ty - #r Pro Glu Gly Ser Lys           # 75                                                                          - CCA GTG AAG GTG CCT GAG AAC CCA CCC TCC AT - #G GTC TTT AAG CAG ATG          349                                                                          Pro Val Lys Val Pro Glu Asn Pro Pro Ser Me - #t Val Phe Lys Gln Met           #                 90                                                          - GAA CAG GTG GCT CAA TTC TTG AAG GCA GCT GA - #A GAT TAT GGA GTC ATC          397                                                                          Glu Gln Val Ala Gln Phe Leu Lys Ala Ala Gl - #u Asp Tyr Gly Val Ile           #            105                                                              - AAG ACT GAC ATG TTC CAG ACT GTT GAC CTC TA - #T GAA GGT AAG GAT ATG          445                                                                          Lys Thr Asp Met Phe Gln Thr Val Asp Leu Ty - #r Glu Gly Lys Asp Met           #       120                                                                   - GCA GCA GTG CAG AGG ACT CTA ATG GCT TTG GG - #C AGT TTG GCT GTG ACC          493                                                                          Ala Ala Val Gln Arg Thr Leu Met Ala Leu Gl - #y Ser Leu Ala Val Thr           #   135                                                                       - AAA AAC GAT GGA AAC TAC CGT GGA GAT CCC AA - #C TGG TTT ATG AAG AAA          541                                                                          Lys Asn Asp Gly Asn Tyr Arg Gly Asp Pro As - #n Trp Phe Met Lys Lys           140                 1 - #45                 1 - #50                 1 -       #55                                                                           - GCC CAG GAG CAT AAG AGG GAC TTC ACA GAC AG - #C CAA CTG CAG GAG GGG          589                                                                          Ala Gln Glu His Lys Arg Asp Phe Thr Asp Se - #r Gln Leu Gln Glu Gly           #               170                                                           - AAG CAC GTC ATT GGC CTT CAA ATG GGC AGC AA - #C AGA GGA GCC TCG CAG          637                                                                          Lys His Val Ile Gly Leu Gln Met Gly Ser As - #n Arg Gly Ala Ser Gln           #           185                                                               - GCT GGC ATG ACA GGC TAT GGG CGA CCC CGG CA - #G ATC ATC AGT TA              #681                                                                          Ala Gly Met Thr Gly Tyr Gly Arg Pro Arg Gl - #n Ile Ile Ser                   #       200                                                                   - GAAAGGGAAG GCCAGCCCTG AGCTGCAGCA TCCTGCTTAG CCTGCCTCAC AA - #ATGCCTAT        741                                                                          - GTAGGTTCTT AGCCCTGACA GCTCTGAGGT GTCACTGGGC AAAGATGACT GC - #ACATGGGC        801                                                                          - AGCTCCCACC TATCCTTAGC CTCAGCCCAG CATCTTACCC CAGAGCCACC AC - #TGCCCTGG        861                                                                          - CCCCTGTTCC CAGCTGTACC CCCACCTCTA CTGTTCCTCT CATCCTGGAG TA - #AGCAGGGA        921                                                                          - GAAGTGGGCT GGGGTAGCTG GCTGTAGGCC AGCCCACTGT CCTTGATATC GA - #ATGTCCTT        981                                                                          - TGAAGGAGAC CCAGCCCAGC CTCTACATCT TTTCCTGGAA TATGTTTTTG GG - #TTGAAATT       1041                                                                          - CAAAAAGGAA AAAAGAAAAA TATATAAATA TATATATATA CAAAAAAAAA AA - #AAAAAAAA       1101                                                                          #             1102                                                            - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 201 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Met Ala Asn Lys Gly Pro Ser Tyr Gly Met Se - #r Arg Glu Val Gln Ser         #                 15                                                          - Lys Ile Glu Lys Lys Tyr Asp Glu Glu Leu Gl - #u Glu Arg Leu Val Glu         #             30                                                              - Trp Ile Val Val Gln Cys Gly Pro Asp Val Gl - #y Arg Pro Asp Arg Gly         #         45                                                                  - Arg Leu Gly Phe Gln Val Trp Leu Lys Asn Gl - #y Val Ile Leu Ser Lys         #     60                                                                      - Leu Val Asn Ser Leu Tyr Pro Glu Gly Ser Ly - #s Pro Val Lys Val Pro         # 80                                                                          - Glu Asn Pro Pro Ser Met Val Phe Lys Gln Me - #t Glu Gln Val Ala Gln         #                 95                                                          - Phe Leu Lys Ala Ala Glu Asp Tyr Gly Val Il - #e Lys Thr Asp Met Phe         #           110                                                               - Gln Thr Val Asp Leu Tyr Glu Gly Lys Asp Me - #t Ala Ala Val Gln Arg         #       125                                                                   - Thr Leu Met Ala Leu Gly Ser Leu Ala Val Th - #r Lys Asn Asp Gly Asn         #   140                                                                       - Tyr Arg Gly Asp Pro Asn Trp Phe Met Lys Ly - #s Ala Gln Glu His Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Asp Phe Thr Asp Ser Gln Leu Gln Glu Gl - #y Lys His Val Ile Gly         #               175                                                           - Leu Gln Met Gly Ser Asn Arg Gly Ala Ser Gl - #n Ala Gly Met Thr Gly         #           190                                                               - Tyr Gly Arg Pro Arg Gln Ile Ile Ser                                         #       200                                                                   - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                # 43               CTCC TTCCAGCCCA CAAACGACCA AGC                             - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #         33       GCTG CCCATGTGCA GTC                                        - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #               25 CTTG TTGGC                                                 - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 1                                                     #/mod.sub.-- base= OTHERRMATION:                                              #"Y = C or T/U"/note=                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 4                                                     #/mod.sub.-- base= OTHERRMATION:                                              #"W = A or T/U"/note=                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 10                                                    #/mod.sub.-- base= OTHERRMATION:                                              #"R = A or G"  /note=                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Cys Cys Cys Met Asn Ser Ser Ser                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Lys Arg Gly Gly Cys Lys Arg Arg Lys                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Thr Lys Asn Asn Gly Asn Ala Ala Lys                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Met Ile Arg Ile Cys Arg Lys Lys                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 381 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - AGTCAAGACT AGTTCCCACC AACTCGATTT TAAAGCCTTG CAAGAAGGTG GC - #TTGTTTGT         60                                                                          - CCCTTGCAGG TTCCTTTGTC GGGCCAAACT CTAGAATGCC TCCCCCTTTC TT - #TCTCATTG        120                                                                          - AAGAGCAGAC CCAAGTCCGG GTAACAAGGA AGGGTTTCAG GGTCCTGCCC AT - #AAAAGGTT        180                                                                          - TTTCCCGGCC GCCCTCAGCA CCGCCCCGCC CCGACCCCCG CAGCATCTCC AA - #AGCATGCA        240                                                                          - GAGAATGTCT CCGGCTGCCC CCGACAGACT GCTCCAACTT GGTGTCTTTC CC - #CAAATATG        300                                                                          - GAGCCTGTGT GGAGTGAGTG GGGCGGCCCG GGGTGGTGAG CCAAGCAGAC TT - #CCATGGGC        360                                                                          #                 381AC G                                                     - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #                47GTCC TGCCCATAAA AGGTTTTTCC CGGCCGC                         - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #                47GTCC TGCCCATAGA TCTTTTTTCC CGGCCGC                         - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                # 43               CCCG CCCCGAGGCC CGCAGCATGT CCG                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                # 43               GGAT CCCCGACCCC CGCAGCATCT CCG                             - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #      37          GAAT GTCTCCGGCT GCCCCCG                                    - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #      37          CAAT GAATTCGGCT GCCCCCG                                    - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                # 44               TCCC CAAATATGGA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                # 44               TCCC CAAGGATCCA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                # 44               TCCC CGGATATGGA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                # 44               TCCC CAAATTAGGA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #21                CAGC G                                                     - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #21                CAGC G                                                     - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #      37          ACAT CACTGCCTAG GCGGCCG                                    - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #      37          CCAT GCCTGCCTAG GCGGCCG                                    - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #      37          CCAT CACTGCCTAG GCGGCCG                                    - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #            29    GGCT TGGCCAGCC                                             - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #              26  GTCA TGGTTA                                                - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #                47GTCC TGCCCATAGA TCTTTTTTCC CGGCCGC                         - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                # 43               GGAT CCCCGACCCC CGCAGCATCT CCG                             - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #      37          CAAT GAATTCGGCT GCCCCCG                                    - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                # 44               TCCC CAAGGATCCA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                # 44               TCCC CGGATATGGA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                # 44               TCCC CAAATTAGGA GCCTGTGTGG AGTG                            - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #21                CAGC G                                                     - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #      37          CCAT GCCTGCCTAG GCGGCCG                                    - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #      37          CCAT CACTGCCTAG GCGGCCG                                    - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 3..8                                                  #/mod.sub.-- base= OTHERRMATION:                                              #"W = A or T"  /note=                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #45                TTCC CCGGATATGG AGCCTGTGTG GAGTG                           - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #45                TTCC CCAAATTAGG AGCCTGTGTG GAGTG                           - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 441 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                - AGTCAAGACT AGTTCCCACC AACTCGATTT TAAAGCCTTG CAAGAAGGTG GC - #TTGTTTGT         60                                                                          - CCCTTGCAGG TTCCTTTGTC GGGCCAAACT CTAGAATGCC TCCCCCTTTC TT - #TCTCATTG        120                                                                          - AAGAGCAGAC CCAAGTCCGG GTAACAAGGA AGGGTTTCAG GGTCCTGCCC AT - #AAAAGGTT        180                                                                          - TTTCCCGGCC GCCCTCAGCA CCGCCCCGCC CCGACCCCCG CAGCATCTCC AA - #AGCATGCA        240                                                                          - GAGAATGTCT CCGGCTGCCC CCGACAGACT GCTCCAACTT GGTGTCTTTC CC - #CAAATATG        300                                                                          - GAGCCTGTGT GGAGTGAGTG GGGCGGCCCG GGGTGGTGAG CCAAGCAGAC TT - #CCATGGGC        360                                                                          - AGGGAGGGGC GCCAGCGGAC GGCAGAGGGG TGACATCACT GCCTAGGCGG CC - #TTTAAACC        420                                                                          #                 441CC A                                                     - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 441 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - TCAGTTCTGA TCAAGGGTGG TTGAGCTAAA ATTTCGGAAC GTTCTTCCAC CG - #AACAAACA         60                                                                          - GGGAACGTCC AAGGAAACAG CCCGGTTTGA GATCTTACGG AGGGGGAAAG AA - #AGAGTAAC        120                                                                          - TTCTCGTCTG GGTTCAGGCC CATTGTTCCT TCCCAAAGTC CCAGGACGGG TA - #TTTTCCAA        180                                                                          - AAAGGGCCGG CGGGAGTCGT GGCGGGGCGG GGCTGGGGGC GTCGTAGAGG TT - #TCGTACGT        240                                                                          - CTCTTACAGA GGCCGACGGG GGCTGTCTGA CGAGGTTGAA CCACAGAAAG GG - #GTTTATAC        300                                                                          - CTCGGACACA CCTCACTCAC CCCGCCGGGC CCCACCACTC GGTTCGTCTG AA - #GGTACCCG        360                                                                          - TCCCTCCCCG CGGTCGCCTG CCGTCTCCCC ACTGTAGTGA CGGATCCGCC GG - #AAATTTGG        420                                                                          #                 441GG T                                                     - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #                47TTCC CACCAACTCG ATTTTAAAGC CTTGCAA                         - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #                47AAGG GTGGTTGAGC TAAAATTTCG GAACGTT                         __________________________________________________________________________

What is claimed is:
 1. A viral vector competent to infect a mammalianhost cell, said vector comprising an SM22α promoter operably linked to anucleotide sequence encoding a polypeptide or RNA other than mouseSM22α, said promoter having a maximum of 441 base pairs andcomprising(a) nucleotides 900-1340 of SEQ ID NO:1, or (b) a sequencehybridizing with the complement of nucleotides 899-1382 of SEQ ID NO:1under hybridizing conditions comprising 0.02M-0.15M sodium chloride attemperatures of 50° C. to 70° C., such that said nucleotide sequenceencoding said polypeptide or RNA is expressed when said vector isprovided to a smooth muscle cell.
 2. The viral of claim 1, wherein saidnucleotide sequence encodes a polypeptide competent to induceangiogenesis.
 3. The viral vector of claim 2, wherein said polypeptidecompetent to induce angiogenesis is a VEGF polypeptide.
 4. The viralvector of claim 1, wherein said nucleotide sequence encodes a cell cycleregulatory molecule.
 5. The viral vector of claim 4, wherein said cellcycle regulatory molecule is a molecule competent to inhibit smoothmuscle cell proliferation.
 6. The viral vector of claim 5, wherein saidcell cycle regulatory molecule is Rb, p53, cell cycle dependent kinase,CDK kinase, or cyclin.
 7. The viral vector of claim 5, wherein said cellcycle regulatory molecule is an RNA antisense molecule.
 8. The viralvector of claim 6, wherein said cell cycle regulatory molecule is aphosphorylation deficient Rb.
 9. The viral vector of claim 6, whereinsaid cell cycle regulatory molecule is a constitutively active Rb cellcycle regulatory molecule.
 10. The viral vector of claim 1, wherein saidviral vector is a raus sarcoma virus vector, a p21 viral vector, aretroviral vector, a herpes simplex viral vector, or a cytomegalovirusvector.
 11. The viral vector of claim 1, wherein said viral vector is anadenoviral vector or an adeno-associated vector.
 12. The viral vector ofclaim 11, wherein said viral vector is replication-deficient.
 13. Theviral vector of claim 1, wherein said smooth muscle cell is an arterialsmooth muscle cell.
 14. The viral vector of claim 1, wherein said smoothmuscle cell is a visceral smooth muscle cell.
 15. An isolated nucleicacid construct comprising an SM22α promoter operably linked to anucleotide sequence encoding a cell cycle regulatory molecule, saidSM22α promoter having a maximum of 441 base pairs and comprising(a)nucleotides 900-1340 of SEQ ID NO:1 or (b) a sequence hybridizing withthe complement of nucleotides 899-1382 of SEQ ID NO:1 under hybridizingconditions comprising 0.02M-0.15 M sodium chloride at temperatures of50° C. to 70° C., such that said cell cycle regulatory molecule isexpressed when said construct is provided to a smooth muscle cell. 16.The construct of claim 15, wherein said cell cycle regulatory moleculeis a molecule competent to inhibit smooth muscle cell proliferation. 17.The construct of claim 16, wherein said cell cycle regulatory moleculeis Rb, p53, cell cycle dependent kinase, CDK kinase, or cyclin.
 18. Theconstruct of claim 16, wherein said cell cycle regulatory molecule is anRNA antisense molecule.
 19. The viral vector of claim 17, wherein saidcell cycle regulatory molecule is a phosphorylation deficient Rb. 20.The construct of claim 17, wherein said cell cycle regulatory moleculeis a constitutively active Rb cell cycle regulatory molecule.
 21. Theconstruct of claim 15, wherein said construct is a viral vector.
 22. Theconstruct of claim 21, wherein said viral vector is a raus sarcoma virusvector, a p21 viral vector, a retroviral vector, a herpes simplex viralvector, or a cytomegalovirus vector.
 23. The construct of claim 21,wherein said viral vector is an adenoviral vector or an adeno-associatedvector.
 24. The construct of claim 23, wherein said viral vector isreplication-deficient.
 25. The construct of claim 15, wherein saidsmooth muscle cell is an arterial smooth muscle cell.
 26. The constructof claim 15, wherein said smooth muscle cell is a visceral smooth musclecell.
 27. A viral vector competent to infect a mammalian host cell, saidvector comprising an SM22α promoter operably linked to a nucleotidesequence encoding a polypeptide or RNA other than mouse SM22α, saidpromoter having a maximum of 441 base pairs and comprising(a) anoligomer of at least one DNA sequence selected from the group consistingof SME-1 (SEQ ID NO:21), SME-2 (SEQ ID NO:23), SME-3 (SEQ ID NO:25),SME4 (SEQ ID NO:27), SME-5 (SEQ ID NO:31), and SME-6 (SEQ ID NO:33), or(b) a sequence that hybridizes with the complement of at least one DNAsequence selected from the group consisting of: SME-1 (SEQ ID NO:21),SME-2 (SEQ ID NO:23), SME-3 (SEQ ID NO:25), SME-4 (SEQ ID NO:27), SME-5(SEQ ID NO:31), and SME-6 (SEQ ID NO:33) under hybridizing conditionscomprising 0.02M-0.15M sodium chloride at temperatures of 50° C. to 70°C., such that said nucleotide sequence encoding said polypeptide or RNAis expressed when said viral vector is provided to a smooth muscle cell.28. The viral vector of claim 27, wherein said nucleotide sequenceencodes a polypeptide competent to induce angiogenesis.
 29. The viralvector of claim 28, wherein said polypeptide competent to induceangiogenesis is a VEGF polypeptide.
 30. The viral vector of claim 27,wherein said nucleotide sequence encodes a cell cycle regulatorymolecule.
 31. The viral vector of claim 30, wherein said cell cycleregulatory molecule is a molecule competent to inhibit smooth musclecell proliferation.
 32. The viral vector of claim 31, wherein said cellcycle regulatory molecule is Rb, p53, cell cycle dependent kinase, CDKkinase, or cyclin.
 33. The viral vector of claim 31, wherein said cellcycle regulatory molecule is an RNA antisense molecule.
 34. The viralvector of claim 32, wherein said cell cycle regulatory molecule is aphosphorylation deficient Rb.
 35. The viral vector of claim 32, whereinsaid cell cycle regulatory molecule is a constitutively active Rb cellcycle regulatory molecule.
 36. The viral vector of claim 27, whereinsaid viral vector is a raus sarcoma virus vector, a p21 viral vector, aretroviral vector, a herpes simplex viral vector, or a cytomegalovirusvector.
 37. The viral vector of claim 27, wherein said viral vector isan adenoviral vector or an adeno-associated vector.
 38. The viral vectorof claim 37, wherein said viral vector is replication-deficient.
 39. Theviral vector of claim 27, wherein said smooth muscle cell is an arterialsmooth muscle cell.
 40. The viral vector of claim 27, wherein saidsmooth muscle cell is a visceral smooth muscle cell.
 41. An isolatednucleic acid construct comprising an SM22α promoter operably linked to anucleotide sequence encoding a muscle contraction inhibiting molecule,said promoter having a maximum of 441 base pairs and comprising(a)nucleotides 900-1340 of SEQ ID NO:1, or (b) a sequence hybridizing withthe complement of nucleotides 899-1382 of SEQ ID NO:1 under hybridizingconditions comprising 0.02M-0.15M sodium chloride at temperatures of 50°C. to 70° C., such that said nucleotide sequence encoding said musclecontraction inhibiting molecule is expressed when said vector isprovided to a smooth muscle cell.
 42. The construct of claim 41, whereinsaid muscle contraction inhibiting molecule comprises the peptideMIRICRKK (SEQ ID NO:19).
 43. An isolated nucleic acid constructcomprising an SM22α promoter operably linked to a nucleotide sequenceencoding a cell cycle regulatory molecule, said promoter having amaximum of 441 base pairs and comprising(a) an oligomer of at least oneDNA sequence selected from the group consisting of SME-1 (SEQ ID NO:21),SME-2 (SEQ ID NO:23), SME-3 (SEQ ID NO:25), SME-4 (SEQ ID NO:27),SME-(SEQ ID NO:31), and SME-6 (SEQ ID NO:33), or (b) a sequence thathybridizes with the complement of at least one DNA sequence selectedfrom the group consisting of: SME-1 (SEQ ID NO:21), SME-2 (SEQ IDNO:23), SME-3 (SEQ ID NO:25), SME-4 (SEQ ID NO:27), SME-5 (SEQ IDNO:31), and SME-6 (SEQ ID NO:33) under hybridizing conditions comprising0.02M-0.15 M sodium chloride at temperatures of 50° C. to 70° C., suchthat said nucleotide sequence encoding said cell cycle regulatorymolecule is expressed when said construct is provided to a smooth musclecell.
 44. The construct of claim 43, wherein said cell cycle regulatorymolecule is a molecule competent to inhibit smooth muscle cellproliferation.
 45. The construct of claim 44, wherein said cell cycleregulatory molecule is Rb, p53, cell cycle dependent kinase, CDK kinase,or cyclin.
 46. The construct of claim 44, wherein said cell cycleregulatory molecule is an RNA antisense molecule.
 47. The viral vectorof claim 45, wherein said cell cycle regulatory molecule is aphosphorylation deficient Rb.
 48. The construct of claim 45, whereinsaid cell cycle regulatory molecule is a constitutively active Rb cellcycle regulatory molecule.
 49. The construct of claim 43, wherein saidconstruct is a viral vector.
 50. The construct of claim 49, wherein saidviral vector is a raus sarcoma virus vector, a p21 viral vector, aretroviral vector, a herpes simplex viral vector, or a cytomegalovirusvector.
 51. The construct of claim 49, wherein said viral vector is anadenoviral vector or an adeno-associated vector.
 52. The construct ofclaim 51, wherein said viral vector is replication-deficient.
 53. Theconstruct of claim 43, wherein said smooth muscle cell is an arterialsmooth muscle cell.
 54. The construct of claim 43, wherein said smoothmuscle cell is a visceral smooth muscle cell.
 55. An isolated nucleicacid construct comprising an SM22α promoter operably linked to anucleotide sequence encoding a polypeptide competent to induceangiogenesis, said promoter having a maximum of 441 base pairs andcomprising(a) an oligomer of at least one DNA sequence selected from thegroup consisting of SME-I (SEQ ID NO:21), SME-2 (SEQ ID NO:23), SME-3(SEQ ID NO:25), SME-4 (SEQ ID NO:27), SME-5 (SEQ ID NO:31), and SME-6(SEQ ID NO:33), or (b) a sequence that hybridizes with the complement ofat least one DNA sequence selected from the group consisting of: SME-1(SEQ ID NO:21), SME-2 (SEQ ID NO:23), SME-3 (SEQ ID NO:25), SME-4 (SEQID NO:27), SME-5 (SEQ ID NO:31), and SME-6 (SEQ ID NO:33) underhybridizing conditions comprising 0.02M-0.15M sodium chloride attemperatures of 50° C. to 70° C., such that said nucleotide sequenceencoding said polypeptide competent to induce angiogenesis is expressedwhen said construct is provided to a smooth muscle cell.
 56. Theconstruct of claim 55, wherein said polypeptide competent to induceangiogenesis is a VEGF polypeptide.
 57. The construct of claim 55,wherein said construct is a viral vector.
 58. The construct of claim 57,wherein said viral vector is a raus sarcoma virus vector, a p21 viralvector, a retroviral vector, a herpes simplex viral vector, or acytomegalovirus vector.
 59. The construct of claim 57, wherein saidviral vector is an adenoviral vector or an adeno-associated vector. 60.The construct of claim 59, wherein said viral vector isreplication-deficient.
 61. The construct of claim 55, wherein saidsmooth muscle cell is an arterial smooth muscle cell.
 62. The constructof claim 55, wherein said smooth muscle cell is a visceral smooth musclecell.